Antibody selective for a tumor necrosis factor-related apoptosis-inducing ligand receptor and uses thereof

ABSTRACT

An antibody of the invention interacts with human DR5 to produce agonistic or antagonistic effects downstream of the receptor including inhibition of cell proliferation and apoptosis. Nucleic acid sequences and amino acid sequences of anti-DR5 antibodies have been elucidated and vectors and cells containing and expressing these sequences have been generated. Methods and uses for the antibodies are detailed including treatment of apoptosis-related disease and treatment of dysregulated cell growth.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of and claims priority to U.S. Ser.No. 13/269,811, filed Oct. 10, 2011, which is currently pending andwhich is a continuation of U.S. Ser. No. 12/822,732, filed Jun. 24,2010, now U.S. Pat. No. 8,067,001, which is a continuation of U.S. Ser.No. 11/760,491, filed Jun. 8, 2007, now U.S. Pat. No. 7,790,165, whichis a continuation of U.S. Ser. No. 10/275,180, filed Mar. 5, 2003, nowU.S. Pat. No. 7,244,429, which is a §371 of International ApplicationNo. PCT/US2001/14151 filed May 2, 2001, now expired. InternationalApplication No. PCT/US2001/14151 claims priority to U.S. Ser. No.60/201,344 filed May 2, 2000. The applications to which the presentapplication claims benefit are herein incorporated by reference in theirentireties.

FIELD OF THE INVENTION

The present invention relates to an antibody capable of specificallybinding a single type of tumor necrosis factor (hereinafter referred toas “TNF”)-related apoptosis-inducing ligand (hereinafter referred to as“TRAIL”) receptor, more particularly, to a monoclonal antibody thatinduces apoptosis in in vivo and in vitro cells expressing the singletype receptor and therapies based thereon.

BACKGROUND OF THE INVENTION

TRAIL is a member of the TNF family of proteins, which also includesTNF-α and Fas ligand (1). These proteins are potent inducers ofapoptosis. To date, five receptors for TRAIL have been identified, twoof which, DR4 (TRAIL-R1) and DR5 (TRAIL-R2) (2-7), are capable oftransducing the apoptosis signal while the other three DcR1 (TRAIL-R3),DcR2 (TRAIL-R4), and osteoprotegerin (OPG) do not transduce theapoptosis signal (8-12). All five receptors for TRAIL share significanthomology in their extracellular ligand binding domains. Similar to Fasand TNF receptor 1 (hereinafter referred to as “TNFR1”), theintracellular segments of both DR4 and DR5 contain a death domain, andtransduce an apoptosis signal through a pathway that involves theFas-associated death domain protein (hereinafter referred to as “FADD”)and caspase 8 (6,7). In addition to transducing the apoptosis signal,the DR4 and DR5 receptors can also activate a pathway involving NFκb(6,7).

The biological functions of TRAIL that have been demonstrated includethe capability of TRAIL to selectively induce apoptosis of transformedtumor cells, with normal cells being relatively resistant toTRAIL-mediated apoptosis (13-15). This selectivity suggests that, incontrast to Fas ligand, the administration of TRAIL is associated withvery low levels of toxicity as demonstrated by systemic administrationof TRAIL in an animal model without inducing significant toxicity (13).Thus, TRAIL has been proposed as a potent apoptosis inducing agent thatwould be a suitable therapeutic agent for the treatment of cancer andother diseases associated with abnormal cell proliferation. TRAIL alsohas been proposed to be a potent apoptosis-inducing agent that would besuitable for the treatment of autoimmune and inflammatory diseases. Ithas been demonstrated that TRAIL-mediated apoptosis is involved inactivation-induced cell death of T cells, thereby serving as analternative mechanism to Fas ligand (16,17). TRAIL-mediated apoptosismay also function in the induction of apoptosis of T cells and otherinflammatory cells (18), and plays a role in the killing activity of NKcells (19-21), and in the immunomodulatory function of dendritic cells(22,23). Thus, TRAIL-mediated apoptosis may also function inimmunoprivilege and immunosurveillance.

The TRAIL receptor system is complex, and includes at least two deathreceptors, DR4 and DR5, and at least two non-apoptotic receptors. DcR1and DcR2. All of these receptors not only share a high amino acidsequence homology, but also exhibit a similar binding affinity to TRAIL(2-12). The ability of the DcR1 and DcR2 receptors to compete forbinding of TRAIL without inducing apoptosis suggests that they may actas decoy receptors that block or modulate the activity of the TRAILligand. Moreover, it has been reported that untransformed cells expresshigher levels of decoy receptors than do transformed cells. Thus, it hasbeen proposed that the differential modulation of the expression of thedeath and decoy receptors may represent a key regulatory mechanism thatdetermines the susceptibility of cells to TRAIL-mediated apoptosis, butdue to the lack of receptor-specific antibodies (2). Although theexpression and function of DR4 and DR5 have been studied extensively,progress has been impeded by the lack of receptor-specific monoclonalantibodies. The cell surface expression of DR5 has not been documented.It has been reported that a panel of anti-TRAIL receptor antibodies havebeen generated that are capable of inducing apoptosis of melanoma cellsin vitro but only upon immobilization of the antibodies, to promotecross-linking, and, in some cases, the cells require culturing withactinomycin D (24). Several anti-DR5 antibodies have been generated(24). However, these previously generated anti-DR5 monoclonal antibodieshave low apoptosis-inducing activity in vitro, even under the conditionsof crosslinking. No in vivo activity has been reported. These antibodieshave not been used for examining cell surface expression of TRAILreceptors (24). Thus, there exists a need for a monoclonal antibodyselective for each specific TRAIL receptor that is not only able to bindto cell surface receptor but also to strongly induce apoptosis ofvarious types of abnormal cells, including tumor cells, both in vivo andvitro without the requirement for crosslinking or immobilization. Suchan antibody would not only provide potential therapeutic agent but alsoa diagnostic tool for functional analysis of TRAIL receptor. Thereexists a particular need for an antibody specific against each of thedeath inducing receptors DR4 and DR5.

In the development, or progression, of many diseases it is often thecase that cells are not deleted. In many autoimmune diseases andinflammatory conditions, the surviving activated cells attack normaltissues or cells. Further, progression of tumorigenesis and theproliferative panus formation of rheumatoid arthritis are characterizedby the unchecked proliferation of cells. Thus, insufficient apoptosisleads to the development of disease, and the uses of apoptosis-inducingligand or agonistic monoclonal antibody to enhance apoptosis areconsidered as a potential therapeutic strategy for eliminating thoseunwanted cells.

For example, rheumatoid arthritis (hereinafter referred to as “RA”) is acommon human autoimmune disease. The current understanding of thepathophysiology of RA is that autoimmune T cells and B cells initiate aninflammatory response in the joints, which drives hyperproliferation ofthe synoviocytes. As a consequence of the hyperproliferation of synovialcells, metalloproteinases (hereinafter referred to as “MMPs”) areover-produced, which further leads to the erosive destruction of thecartilage and bone that is characteristic of RA (25). Thus, the controlof hyperproliferation of inflammatory synovial cells is a key step inthe treatment of RA. The molecular mechanisms leading to thehyperproliferation of synovial cells are still unknown. Although thehyperproliferative synovial cells are non-malignant and non-transformed,many studies have suggested that they share some common features withtransformed cells (46). These cells, the so-called,“transformed-appearing synoviocytes”, are characterized by a dense roughendoplasmic reticulum, numerous irregular nuclei, and changes in thenormally spindle-shaped cell skeleton. It has been proposed that theincorporation of the oncogenes and virus-derived genes might be theprimary triggers for the transformed appearance of RA synovial cells(46).

At least two aspects of RA suggest that dysregulated apoptosis maycontribute to the disease process and that therapeutic elicitation ofapoptosis may be an effective treatment: the failure of the deletion ofthe activated T cells suggests that there is defectiveactivation-induced cell death of these T cells, which is a process thatinvolves Fas-mediated apoptosis and TRAIL-mediated apoptosis, and thehyperproliferative nature of the RA synovial cells is a contributingfactor in the later stages of RA pathophysiology. Indeed, it has beenshown that the administration of anti-Fas antibody into the inflammatoryjoint inhibits the development of chronic arthritis in tax transgenicmice, which are an animal model for human RA (26). Moreover, localizedtransduction with the fas ligand gene by an adenoviral vector iseffective in prevention of collagen-induced arthritis (27). Inhibitionof the proliferation of inflammatory synovial cells by enhancement ofFas-mediated apoptosis is observed in both cases. Although Fas ligand isa strong apoptosis inducer in RA synovial cells, the application of Fasligand-mediated apoptosis as a therapy for humans has been limited bylethal liver toxicity. Thus, TRAIL receptor induced apoptosis representsa safer and more effective therapeutic for the treatment of RA thanFas-ligand induced apoptosis.

TRAIL receptor induced apoptosis also represents a safer and moreeffective therapeutic for the treatment of cancer than Fas-ligandinduced apoptosis. TRAIL-mediated apoptosis is known to specificallyinduce apoptosis of transformed tumor cells without affecting normalcells. It has been shown that the systemic administration of thetrimerized soluble TRAIL did not cause toxicity in experimental animalsyet was able to induce regression of implanted tumors (13,28). Itspotential as an adjunctive therapy for traditional treatments wasunderscored by the recent finding that the expression of DR5 andsusceptibility to TRAIL-induced apoptosis of breast cancer cells isenhanced by the radiation, suggesting that combined with radiation, theefficiency of TRAIL would be increased in cancer therapy (29).

In addition, the gene encoding the TRAIL receptor DR5 has been mapped tochromosome 8p21-22, loci with a high frequency of mutation in somecancer cells (30). It has been reported that at least two kinds of tumorcells, small lung cancer (31) and head and neck cancer (32) exhibitmutations in the death domain of the DR5 gene. Thus, there exists a needfor an anti-DR5 antibody in cancer research to determine the effect ofreceptor epitope variation on the development and progression ofcancers. Further, the functionality of TRAIL receptor mutations wouldprove a useful clinical diagnostic tool when used in conjunction withother biomarkers in the early detection of cancers and as a predictor ofthe tumor aggressiveness.

SUMMARY OF THE INVENTION

An antibody is disclosed which recognizes a TRAIL receptor DR5 and whichinduces apoptosis in a DR5-expressing cell in vivo. Further disclosed isan antibody that recognizes DR5 but not DR4, DcR1, or DcR2. Specificallydetailed is a monoclonal antibody to DR5 produced by a hybridoma.

A method provided by the invention allows inhibition of cellproliferation by exposing a cell to a therapeutic quantity of anantibody capable of binding to DR5. Also disclosed is a pharmacologicalcomposition that includes a therapeutic amount of monoclonal antibodyactive against a DR5, a pharmaceutically acceptable carrier and acontainer enclosing the antibody and the carrier. Further provided bythe invention is the use of an antibody recognizing DR5 for preparing atherapeutic for selective apoptosis of abnormal or dysregulated cells.

An antibody of the present invention interacts with a tumor necrosisfactor ligand receptor such as DR4, DR5, DrR1, DrR2 and OPG, inducingapoptosis in a cell expressing such a receptor. Disclosed is an antibodyof the invention capable of selectively binding an agonistic orantagonistic tumor necrosis factor ligand receptor epitope.

The present invention provides a treatment for an apoptosis relateddisease by a method that includes exposing a target tissue having anapoptosis related disease to a therapeutic quantity of an antibody ofthe invention.

Further described is a fusion protein that includes an antigenic TRAILreceptor amino acid sequence having at least ten bases, coupled to animmunoglobulin protein or fragment thereof capable of eliciting animmune response within a subject.

The present invention provides a method of gene therapy in which atarget cell is transfected with a TRAIL receptor nucleic acid sequencein an expression vector so that the TRAIL receptor is expressed on thetarget cell. The target cell is then exposed to an antibody thatselectively binds the TRAIL receptor.

Provided are nucleic acid sequences and amino acid sequences encodingthe heavy and light chain immunoglobulins of an antibody selective forDR5. Also detailed are vectors that include a nucleic acid sequence ofthe invention and host cells transformed with a vector of the invention.

The present invention provides a host cell producing a humanized TRA-8.

A process for producing a humanized DR5 antibody is described in which ahost is transformed with nucleic acid sequences encoding a humanizedimmunoglobulin light chain and a humanized immunoglobulin heavy chainafter which the transformed host is incubated for a predetermined periodof time.

Also described is a process for inhibiting cell proliferation thatincludes contacting a target cell with a pharmaceutically effectiveamount of a humanized DR5 antibody.

A commercial kit is provided for inducing apoptosis that includes ahumanized TRA-8 antibody selective for DR5; packaged in a suitablecontainer together with instructions for use.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B-1, 1B-2, 1B-3, 1C, 1D1, 1D2, 1E, 1F, and 1G showcharacterization of TRA-8. FIG. 1A shows binding specificity of TRA-8:Western blot analysis (upper panel): Recombinant fusion proteins of theTNFR family probed with TRA-8 or anti-human IgG. Lane 1: DR5/hIgG1fusion protein (immunogen); Lane 2: DR4/hIgG1 (TRAIL-R1); Lane 3:DR5/hIgG1; Lane 4: TRAIL-R3 (DcR-1)/hIgG1; Lane 5: TRAIL-R4(DcR-2)/hIgG1; Lane 6, CD95/hIgG1; Lane 7: soluble TNFR1. ELISA analysis(lower panel): The well numbers match those of the Western blot exceptwell 8 which is a murine DR5/hIgG1 fusion protein. FIGS. 1B-1, 1B-2 and1B-3 show binding activity of soluble TRAIL and TRA-8 to DR5 and DR4:ELISA plates were coated with DR5/hIgG1 or DR4/hIgG1 and then incubatedwith TRAIL or TRA-8. FIG. 1C shows Flow cytometry analysis of thesurface expression of DR5. Cos-7 cells transfected with pcDNA3expression vector containing the full-length DR5 cDNA, DR4 cDNA or emptyvector. Forty-eight hours after transfection, cells were stained withTRA-8 followed by PE-conjugated anti-mouse IgG1. FIGS. 1D-1 and 1D-2show in situ immunohistochemistry reactivity for DR5: Cytospin slides ofCos-7 cells transfected with DR5 expression or control vector werestained with TRA-8 at 48 hours after transfection. FIG. 1E shows killingactivity of TRA-8: Jurkat cells were incubated with the indicatedconcentrations of TRA-8. Cell viability was determined by ATPLite®, MTT,and PI exclusion assays after overnight culture. The results of ATPLite®and MTT assays are presented as percent of medium control, and PI assayare presented as percent of PI negative cells. FIG. 1F shows Westernblot analysis of caspase activation: Jurkat cells were incubated with500 ng/ml TRA-8 for indicated time. Cell lysates were separated by 15%SDS-PAGE, blotted, and probed with anti-caspase antibodies. The arrowsindicate the cleaved subunits of each caspase. FIG. 1G shows caspaseinhibition assay: Jurkat cells were incubated with 50 ng/ml TRA-8overnight in the presence of various concentrations of indicated caspaseinhibitors. Cell viability was determined by the ATPLite® assay.

FIGS. 2A-1, 2A-2, 2A-3, 2A-4, 2A-5, 2A-6, 2A-7, 2A-8, 2A-9, 2A-10, 2A′,2B-1, 2-B2, 2-B3, 2-B4, 2-B5, 2-B6, 2-B7, 2-B8, 2-B9, 2B-10, 2B′-1,2B′-2, 2B′-3, 2B′-4, 2B′-5, 2B′-6, 2B′-7, 2B′-8, 2B∝-9, 2B∝-10, 2C-1,2C-2, 2C-3, 2D-1, 2D-2, 2D-3, 2D-4, 2D-5, 2D-6, 2D-7, 2D-8, 2D-9 to2D-10 show cell surface expression of DR5 and susceptibility toDR5-mediated apoptosis. Normal T and B cells, freshly isolated fromperipheral blood, T cell (FIGS. 2A-1 to 2A-10 and 2A′), glioma (FIGS.2B-1 to 2B-10 and 2B′-1 to 2B′-10), prostate cancer cell (FIGS. 2C-1 to2C-3) and B cell (FIGS. 2D-1 to 2D-10) cell lines were incubated withTRA-8 or murine IgG isotype control antibody followed by PE-conjugatedgoat anti-mouse IgG1. Apoptosis was determined by the ATPLite® assayafter overnight incubation with soluble TRAIL (open circles) or TRA-8(closed circles) as shown in FIGS. 2A-6 to 2A-10, 2B′-6 to 2B′-10 and2D-6 to 2D-10.

FIG. 3A′ shows T cell line U937 was incubated with TRA-8 or murine IgG1isotype control antibody. Apoptosis was determined by the ATPLite® assayafter overnight incubation with soluble TRAIL (open circles) or TRA-8(closed circles).

FIGS. 3B-1, 3B-2, 3B-3, 3B-4, 3B-5, 3B-6, 3B-7, 3B-8, 3B-9, 3B-10, 3C-1,3-2, and 3C-3 show glioma (FIGS. 3B-1 to 3B-10) and prostate cancer(FIGS. 3C-1 to 3C-3) cell lines were incubated with TRA-8 or murine IgG1isotype control antibody. Apoptosis was determined by the ATPLite® assayafter overnight incubation with soluble TRAIL (open circles) or TRA-8(closed circles).

FIGS. 4A and 4B are a series of graphs showing cell viability for humanJurkat cells after exposure to indicated concentrations of (FIG. 4A)antibody strains TRA-1, -8 and -10 and (FIG. 4B) TRAIL in the presenceof a fixed concentration of the inventive antibody strains depicted inFIG. 4A.

FIGS. 5A, 5B, 5C and 5D show expression of DR5 in normal and cancertissues: Normal and cancer tissue homogenates were probed with TRA-8 anddeveloped by chemiluminescence. FIG. 5A shows Western blot analysis ofDR5 protein in normal tissues: lane 1: liver, lane 2: brain, lane 3:lung, lane 4: kidney, lane 5: spleen, lane 6: testes, Lane 7: ovary,lane 8: heart, lane 9: pancreas. FIG. 5B shows Western blot analysis ofDR5 protein in cancer tissues. The cancer tissue blot containing cancersfrom the ovary (lane 1), lung (lane 2), liver (lane 3), rectum (lane 4),cervix (lane 5), skin (lane 6), testes (lane 7), thyroid (lane 8),uterus (lane 10), stomach (lane 11), laryngopharynx (lane 12), andpancreas (lane 13) was probed. In situ immunohistochemistry of normalhuman tissues (FIG. 5C) and of cancer tissues (FIG. 5D). Frozen sectionswere immunostained with TRA-8.

FIGS. 6A, 6B, 6C, 6D, and 6E show tumoricidal activity of TRA-8. SCIDmice were inoculated subcutaneously with 1321N1 cells. Mice wereinjected intravenously with a single dose of 100 μg TRA-8 on the secondday after tumor inoculation (FIG. 6A), or with three doses of 100 μgTRA-8 beginning 7 days after tumor inoculation (FIG. 6B). Tumor growthwas determined by the weight and examined histologically with H&Estaining. The photographs show viable tumor growth in control mice butnot in TRA-8 treated mice (FIG. 6C, upper panel), and H&E staining oftumor (FIG. 6C, lower panel). SCID mice were injected intravenously with10⁶ Jurkat cells and treated with a single dose of TRA-8 on the secondday after injection. Seven days later, spleen cells were harvested,stained with anti-human CD3 antibody and analyzed by flow cytometry(FIG. 6D), or by immunohistochemistry (FIG. 6E).

FIGS. 7A-1, 7A-2, 7A-3, 7A-4, 7A-5, 7A-6, 7A-7, 7A-8, 7B-1, 7B-2, 7B-3,7B-4, 7B-5, 7B-6, and 7B-7 show expression of cell surface DR5 in RA(FIGS. 7A-1 to 7A-8) and OA (FIGS. 78-1 to 7B-7) synovial cells. 1×10⁶primary cultured synovial cells were stained with affinity-purifiedTRA-8 and followed by PE-conjugated goat anti-mouse IgG1 antibody.10,000 viable cells analyzed by FACSvantage™.

FIGS. 8A-1, 8A-2, 8A-3, 8A-4, 8A-5, 8A-6, 8A-7, 8A-8, 8B-1, 88-2, 8B-3,and 8B-4 are a series of graphs showing cell viability as a function ofTRAIL and TRA-8 concentration induced apoptosis of representativestrains of RA (FIGS. 8A-1 to 8A-8) and OA (FIGS. 8B-1 to 88-4) synovialcells with various concentrations of the recombinant soluble TRAIL (theopen circles) or affinity-purified TRA-8 (the closed circles). Cellviability is the percentage of the cpm of treated cells versus the cpmof untreated cells.

FIGS. 9A, 9B, 9C, 9D, 9E, 9F, 9G, and 9H are a series of graphs showingthe caspase dependence of DR5-mediated apoptosis of RA synovial cells.RA synovial cells (RA512) are incubated with 50 ng/ml of soluble Fasligand (open squares), anti-Fas antibody (CH-11) (closed squares),soluble TRAIL (open circles), or anti-DR5 antibody (TRA-8) (closedcircles) in the presence of variable concentrations of caspase 1inhibitor (FIG. 9A), caspase 2 inhibitor (FIG. 9B), caspase 3 inhibitor(FIG. 9C), caspase 4 inhibitor (FIG. 9D), caspase 6 inhibitor (FIG. 9E),caspase 8 inhibitor (FIG. 9F), caspase 9 inhibitor (FIG. 90) or caspase10 inhibitor (FIG. 9H). After overnight culture, cell viability isdetermined by ATPLite®.

FIG. 10A is an electrophoretic gel-shift assay indicating NFkbactivation. RA1016 cells are incubated with 20 ng/ml TNF-a, 50 ng/mlsoluble TRAIL, or 50 ng/ml TRA-8 for indicated time points before beingsubjected to electrophoresis. FIGS. 10B and 10C are graphs showing theproduction of MMP-1 and MMP-3. 1×10⁶/ml of indicated RA synovial cellsare incubated with the indicated concentrations of TNF-a (the opencircles), TRAIL (the open triangles) or TRA-8 (the closed circle). Afterovernight culture, the culture supernatants are collected. The levels ofMMPs in culture supernatants are determined by ELISA.

FIGS. 11A and 11B-1, 11-B2, 11-B3, 11-B4, 11-B5, 11-B6, 11-B7, and 11B-8show TRA-8 does not induce hepatocellular toxicity. FIG. 11A showsnormal liver tissues do not express DR5. The paraffin sections of twonormal liver tissues, one hepatocellular carcinoma tissue, and thecytospin preparation of HepG2 cells were prepared for H&E staining, andcorresponding frozen sections were stained with TRA-8. FIG. 11B-1 to11B-8 show flow cytometry analysis of cell surface expression of DR5.Hepatocytes isolated from two normal liver tissues and from a case ofhepatocellular carcinoma tissue, and HepG2 cells were stained withTRA-8, anti-Fas antibody (DX2) or an isotype control antibody. The solidhistograms indicate TRA-8 or DX2 staining, and the open histograms arethe corresponding isotype controls.

FIGS. 12A, 12B, 12C, 12D-1, 12-D2, 12-D3, 12-D4, 12-D5, and 12D-6 showTRAIL but not TRA-8 induces hepatocellular toxicity. Fresh normal humanhepatocytes were maintained in Hepatocyte Culture Medium. FIG. 12A showsapoptosis of hepatocytes was induced with 1 μg/ml soluble TRAIL pluscrosslinker or TRA-8 for the indicated time points. Cell viability wasdetermined by ATPLite®. The results are presented as percent viablecells compared to the medium control. The shaded bars indicate TRAIL andthe black bars indicate TRA-8. FIG. 12B shows the condensed nuclei ofhepatocytes were stained with Hoechst 33352 and analyzed by flowcytometry. FIG. 12C shows the effect of cycloheximide on hepatocytesapoptosis. Hepatocytes were cultured in control medium or with 1 μg/mlTRAIL or TRA-8 in the presence (closed bars) or absence (open bars) of 1μg/ml cycloheximide for 8 hours. Cell viability was determined byATPLite®. The results are presented as mean±SEM of triplicate culturesof two experiments. FIGS. 12D-1 to 12D-6 show comparison of thesusceptibility of normal hepatocytes to DR5 and Fas-mediated apoptosis.Freshly isolated hepatocytes were incubated with indicatedconcentrations of soluble TRAIL, TRA-8, soluble FasL or the anti-Fas mAbCH11 for 6 hours. Cell viability was determined by ATPLite® assay. Theresults are presented as the percentage of viable cells compared tomedium control. For normal hepatocytes, mean±SEM of four normalindividuals are presented. The results of hepatocellular carcinoma cellsfrom one patient and HepG2 cells are presented as the average oftriplicate cultures.

FIGS. 13A, 13B, 13C-1, 13C-2, 13D, and 13E show TRAIL induces hepatitis.B6 mice were intravenously inoculated with 10⁹ pfu of adenoviral vectorencoding the full length of human TRAIL under the control of the“Tet-on” transcriptional element. TRAIL expression was induced by theindicated dose of tetracycline. FIG. 13A shows Northern blot analysis ofhuman TRAIL expression in the liver. 24 hours after inoculation ofvector and induction with tetracycline, total RNA was isolated from thelivers and probed with human TRAIL cDNA or β-actin. FIG. 13B showslevels of AST. 24 hours after transduction of TRAIL, serum levels of ASTwere determined. FIGS. 13C-1 and 13C-2 show TRAIL-mediated cell death ofadenoviral vector infected hepatocytes: B6 mice were intravenouslyinoculated with tetracycline-inducible adenoviral vector. 48 hours afterinoculation, hepatocytes from inoculated and non-inoculated control micewere isolated and incubated with indicated concentrations of TRAIL for 8hours (left panel). Cell viability of hepatocytes was determined by theATPLite® assay. Mice, inoculated with adenoviral vector as above, wereintravenously injected with 10 μg of soluble human TRAIL 48 hours later.Serum levels of AST were measured at 24 hours after TRAIL injection(right panel). FIGS. 13D and 13E show analysis of liver damage inducedby TRAIL. The livers were collected at 24 hours (FIG. 13D) or 7 days(FIG. 13E) after transduction with TRAIL. The paraffin sections were H&Estained, and photographed at 100× (top panel) and 400× (lower panel).

FIGS. 14A, 14B, 14C, 14D, 14E, 14F are a series of graphs showing thatactivated T cells and B cells purified from human PBMC express increasedlevels of DR5 as determined by flow cytometry for resting and activatedcells. FIG. 14A is a graph of DR5 expression in un-stimulated human Tcells. FIG. 14B is a graph of DR5 expression 48 hours after anti-CD3stimulation. FIG. 14C is a graph of DR5 expression 48 hours after Con-Astimulation. FIG. 14D is a graph of DR5 expression in un-stimulated Bcells. FIG. 14E is a graph of DR5 expression after anti-μ stimulation.FIG. 14F is a graph of DR5 expression after LPS expression.

FIGS. 15A and 15B are viability graphs as a function of TRA-8concentration for the purified T cells (FIG. 15A) and B cells (FIG. 15B)depicted in FIG. 14 that have been stimulated for 48 hours with anti-CD3(FIG. 15A) or anti-μ (FIG. 15B), with activated and blast cellscollected by different density of Ficoll-Paque. Viability is determinedby ATPLite® assay.

FIG. 16 is a histogram and flow cytometry plots showing CD3 expressionin a gated lymphocyte population for NK cell depleted NOD/SCID miceinjected with human PBMC and TRA-8 or IgG (control).

FIG. 17 shows CD3 and TUNEL stained cellular micrographs for mousespleen tissue as detailed in Example 13.

FIGS. 18A, 18B and 18C show cycotoxicity plots for chronic lympholyticleukemia (CCL) and normal B cell humans in the presence of TRA-8 (FIG.18A). BISVIII (FIG. 18B), and the combination thereof (FIG. 18C).

DETAILED DESCRIPTION OF THE INVENTION

The failure to delete cells is due to defects in the apoptosis inducingsystem which are associated with defects illustratively includingexpression or function of the ligand, the receptor, or the intracellularregulatory and effector molecules. The present invention affords amethod to correct a deficient apoptosis inducing system as well as toelucidate the specific defects inherent in a given defective apoptosisinducing system.

The present invention relates to a new class of monoclonal antibodiesthat have selective in vive and in vitro apoptosis inducing activityagainst specific TRAIL receptors, including DR5, DR4, DcR1 and DcR2. Thepresent invention has utility as a reagent for apoptosis signalingresearch, as well as a therapeutic effective against cells expressingTRAIL receptors illustratively including broad classes of cancer cells,disregulation of the apoptosis system and abnormally proliferatingsynovial cells of autoimmune diseases. Antibodies according to thepresent invention are specific in binding particular types of TRAILreceptors in spite of the homology therebetween. The inventiveantibodies afford targeted apoptosis of only those cells expressing atarget TRAIL receptor or alternatively, blocking TRAIL apoptosis ofcells expressing a target receptor.

An anti-DR5 monoclonal antibody of the present invention serves as apotent inducer of apoptosis in cells expressing DR5 in vitro and as apotent inducer of apoptosis in vivo. Humanized fragmentary CDR sequencesengrafted on humanized antibody backbones and fusion protein anti-DR5antibodies of the present invention exhibit similar apoptoticproperties.

To date, no monoclonal antibody is available which binds to cell surfaceDR5 and induces apoptosis of cells expressing DR5 both in vitro and invivo in the absence of a crosslinker. The present invention includes aDR5 antibody operative as a therapeutic agent in animal models ofdisease, such as xenografted animals, or in vivo. Although soluble TRAILhas been shown to be effective in induction of apoptosis of tumor cellsin vivo, the killing activity appeared to be very low with the large andrepeated doses often being required (13). TRA-8, one of a series of DR5antibodies according to the present invention, is pharmaceuticallyeffective in animals carrying a human DR5 transgene and also has utilityin establishing a model for the investigation of the role of DR5 andTRAIL.

An antibody according to the present invention raised against a TRAILreceptor is harvested according to the present invention from anexperimental animal. By humanizing the antibody according to the presentinvention to maintain receptor binding activity while eliciting adiminished and therapeutically tolerable immune response within a humansubject, a humanized anti-TRAIL receptor antibody according to thepresent invention is used as therapeutic agonist or antagonist for agiven TRAIL receptor. The present invention being operative as an invivo therapeutic since secondary crosslinking of the anti-TRAIL receptorantibody, optionally, is not required.

The present invention extends beyond a single anti-TRAIL receptorantibody having agonist or antagonistic apoptotic effects. Rather, twoor more anti-TRAIL receptor antibodies are brought into contact with acell culture in vitro or a subject body tissue in vivo to create asynergistic treatment. For example, glioma cell line U87 andhematopoietic cell lines U937 and Molt-4 are responsive to exposure to asynergistic exposure to agonistic anti-DR4 and anti-DR5 antibodieswhereas exposure to agonistic anti-DR5 antibody alone shows only limitedsuccess in inducing apoptosis.

Additionally, antagonistic anti-TRAIL receptor antibodies haveparticular utility in the present invention when an antibody is specificto binding one of the decoy receptors DcR1, DcR2 or OPG. Selectiveblocking of a decoy receptor with an antibody according to the presentinvention has the effect in cell types expressing decoy receptors ofshifting the TRAIL binding equilibrium towards those TRAIL receptorscapable of transducing the apoptosis signal. Thus, in another combinedtherapy according to the present invention, a decoy receptor bindingantibody sensitizes an expressing cell towards agonistic apoptosissignal transducing TRAIL receptor binding.

In another embodiment, the present invention affords a method ofelucidating agonistic and antagonistic epitopes of a given TRAILreceptor. Further, polymorphisms between individuals associated with agiven TRAIL receptor are elucidated according to the present inventionthrough the use of a panel of monoclonal antibodies each having adiffering variable or CDR region. A characterized panel of monoclonalantibodies provides the ability to define agonistic and antagonisticepitopes and polymorphisms. Thus, a panel of monoclonal antibodiesaccording to the present invention has utility in drug discovery and/orsubject screening for disease proclivity.

Still another embodiment of the present invention involves fusionproteins including an antigenic fragment of a TRAIL receptor coupled toan immunoglobulin protein, polypeptide or fragment thereof. A TRAILreceptor fragment being defined as containing a sufficient number ofbases to elicit an immunogenic response to a native TRAIL receptorexpressed on a subject cell surface. A TRAIL receptor fusion fragmentincluding at least ten amino acids. An immunoglobulin fusion protein orfragment thereof is defined herein to include a native or syntheticprotein or polypeptide segment having a sufficient number of amino acidbases to activate an immunogenic cascade response within a subject. Animmunogen of the present invention including a fusion of a TRAILreceptor fragment coupled to an immunoglobin fragment has utility as anin vive therapeutic to elicit an anti-TRAIL receptor antibody in situwithin a subject.

In still a further embodiment, the present invention is operative as agene therapy. In a gene therapy aspect of the present invention,targeted cells are transfected with a vector carrying an expressiblesequence corresponding to a TRAIL receptor. The vector beingconventional and chosen on the basis of the targeted cell susceptibilityto the vector. Gene therapy vectors illustratively include adenovirus,pAdCMV5. Upon the targeted cells or tissue expressing the transfectedTRAIL receptor, the cells or tissue are exposed to an antibody accordingto the present invention specific for binding the transfected TRAILreceptor. It is appreciated that the anti-TRAIL receptor antibody iseither agonistic or antagonistic thereto consistent with the desiredtherapeutic result.

The antibodies of the present invention are also operative inconjunction with a sensitizer. A sensitizer as used herein is defined toinclude any stimulus that induces apoptosis, including ultravioletlight, organic molecules specifically including the class ofbisindolmaleimides, heavy metals and free radical species.

In the context of a malignancy therapy, TRA-8, is able to induceapoptosis of most TRAIL-sensitive tumor cells in a caspase-dependentfashion in the absence of the secondary crosslinking. TRA-8 exhibits astrong tumoricidal activity in vivo. The ability of TRA-8 to induceapoptosis of most TRAIL-sensitive cells confirms that DR5 alone issufficient to trigger apoptosis. The majority of tumor cells detailedherein express cell surface DR5 and their susceptibility to TRA-8induced cell death paralleled their susceptibility to TRAIL, indicatingthat DR5 is a primary death receptor for TRAIL-mediated apoptosis inmost tumor cells. Thus, differential expression of DR5 by normal andcancer cells is operative in the selectivity of TRAIL-mediatedapoptosis. TRA-8 bypasses the decoy receptors to induce TRAIL-mediatedapoptosis. Only a minority of TRAIL, resistant tumor cells are sensitiveto TRA-8, however, indicating that the decoy receptors do not appear toplay a major role in the resistance of tumor cells to TRAIL-mediatedapoptosis.

Although previous studies have indicated that systemic administration ofthe soluble form of TRAIL in animals does induce tumor regressionwithout causing toxicity^(3,4,22), the membrane-bound form of humanTRAIL induces liver damage in mice as shown herein. However, the hepatictoxicity of TRAIL is much less potent than that of Fas ligand asdemonstrated by the lesser susceptibility of normal hepatocytes toTRAIL-induced injury compared to Fas ligand and by the lack of lethalityof TRAIL in vivo. Thus, titration of TRAIL has utility in cancertherapy.

As detailed herein, the absence of significant levels of DR5 proteinexpression by normal hepatocytes is shown and is associated withhepatocyte resistance to TRA-8 induced apoptosis. Crosslinking of DR5with monoclonal antibody is insufficient to organize the homopolymericforms of the death receptor able to trigger apoptosis. Experiments inmarmoset indicate no evidence of hepatic toxicity of TRA-8administration. Thus, an agonistic monoclonal anti-DR5 antibody islikely to be more selective and safer than soluble TRAIL as atherapeutic agent.

As a screening assay, the present invention is well suited for detectingsmall clusters of malignant cells which may still exhibit normal cellmorphology. In situ cell section staining of human cancer cellsincluding lung, prostate and liver cancers with labeled antibodiesaccording to the present invention readily identifies cancerous cells.These cancer cells are observed to express very high levels of DR5 ascompared to normal cells of the same type. Thus, the present inventionhas utility as a sensitive screening method for early stage malignancieswithin tissue including at least lung, prostate and liver. A therapeuticprocess is detailed herein for the inhibition of abnormal cellproliferation associated with diseases illustratively malignant cancersand lymphatic leukemias.

The present invention is detailed herein with particularity to ananti-human DR5 monoclonal antibody designated as TRA-8, having ATCCAccession Number PTA-1428. It is appreciated that the techniques andresults detailed with regard to the agonistic anti-human DR5 monoclonalantibody TRA-8 are wholly extendable and applicable to antagonistic DR5antibodies, as well as antibodies raised against DR4, DcR1 and DcR2acting in both agonistic and antagonistic manners.

The levels of expression of an apoptosis receptor, such as Fas, do notnecessarily correlate with the susceptibility of the cells to apoptosis.For TRAIL-mediated apoptosis, it has been suggested that the expressionof the decoy receptors for TRAIL influences the susceptibility of thecells. Moreover, it has been suggested that DR5 must be associated withDR4 for effective transduction of the apoptosis signal through FADD andthe caspase 8 pathway. The availability of agonistic monoclonal anti-DR5antibody allowed evaluation of the regulation of DR5 signaling and itsrelative role in TRAIL-mediated apoptosis. Comparison of thesusceptibility of the cells to TRA-8-mediated apoptosis with theirsusceptibility to TRAIL-mediated apoptosis offers insight into the roleof DR5 in TRAIL-mediated apoptosis and the mechanisms that may affectsusceptibility.

This advantage generally extends to humanized anti-DR5 antibodies of thepresent invention. A molecular clone of an antibody to DR-5 is preparedby known techniques as detailed with respect to the following Examples.Recombinant DNA methodology (33) is operative herein to constructnucleic acid sequences which encode a monoclonal antibody molecule orantigen binding region thereof.

The present invention allows the construction of humanized anti-TRAILreceptor antibodies that are unlikely to induce a human anti-mouseantibody (hereinafter referred to as “HAMA”) response (34), while stillhaving an effective antibody effector function. As used herein, theterms “human” and “humanized,” in relation to antibodies, relate to anyantibody which is expected to elicit a therapeutically tolerable weakimmunogenic response in a human subject.

The present invention provides for an anti-DR5 antibody, a humanizedanti-DR5 antibody, TRA-8 heavy and light chain immunoglobulins andhumanized heavy and light chain immunoglobulins. Certain truncations ofthese proteins or genes perform the regulatory or enzymatic functions ofthe full sequence protein or gene. For example, the nucleic acidsequences coding therefor can be altered by substitutions, additions,deletions or multimeric expression that provide for functionallyequivalent proteins or genes. Due to the degeneracy of nucleic acidcoding sequences, other sequences which encode substantially the sameamino acid sequences as those of the naturally occurring proteins may beused in the practice of the present invention. These include, but arenot limited to, nucleic acid sequences including all or portions of thenucleic acid sequences encoding the above polypeptides, which arealtered by the substitution of different codons that encode afunctionally equivalent amino acid residue within the sequence, thusproducing a silent change. It is appreciated that the nucleotidesequence of an immunoglobin according to the present invention toleratessequence homology variations of up to 25% as calculated by standardmethods (“Current Methods in Sequence Comparison and Analysis,”Macromolecule Sequencing and Synthesis, Selected Methods andApplications, pp. 127-149, 1998. Alan R. Liss, Inc.) so long as such avariant forms an operative antibody which recognizes a TRAIL receptorDR5. For example, one or more amino acid residues within a polypeptidesequence can be substituted by another amino acid of a similar polaritywhich acts as a functional equivalent, resulting in a silent alteration.Substitutes for an amino acid within the sequence may be selected fromother members of the class to which the amino acid belongs. For example,the nonpolar (hydrophobic) amino acids include alanine, leucine,isoleucine, valine, proline, phenylalanine, tryptophan and methionine.The polar neutral amino acids include glycine, serine, threonine,cysteine, tyrosine, asparagine, and glutamine. The positively charged(basic) amino acids include arginine, lysine and histidine. Thenegatively charged (acidic) amino acids include aspartic acid andglutamic acid. Also included within the scope of the present inventionare proteins or fragments or derivatives thereof which aredifferentially modified during or after translation, e.g., byglycosylation, proteolytic cleavage, linkage to an antibody molecule orother cellular ligands, etc. In addition, the recombinant vectorencoding nucleic acid sequences of the anti-DR5 antibodies of thepresent invention may be engineered so as to modify processing orexpression of a vector.

Additionally, an inhibitor encoding nucleic acid sequence can be mutatedin vitro or in vivo to create and/or destroy translation, initiation,and/or termination sequences or to create variations in coding regionsand/or form new restriction endonuclease sites or destroy pre-existingones, to facilitate further in vitro modification. Any technique formutagenesis known in the art can be used, including but not limited toin vitro site directed mutagenesis, J. Biol. Chem. 253:6551, use of Tablinkers (Pharmacia), and the like.

X-ray crystallography data indicate that the antibody immunoglobulinfold generally forms a long cylindrical structure comprising two layersof antiparallel b-sheets, each consisting of three or four b-chains. Ina variable region, three loops from each of the V domains of H and Lchains cluster together to form an antigen-binding site. Each of theseloops is termed a complementarity determining region (CDR). The CDRshave the highest variability in amino acid sequence with the antibody.The portions of the variable region that are not part of a CDR arecalled “framework regions” (“FR” regions) and generally play a role inmaintaining CDR structure. Preferably, all the CDRs from a givenantibody are grafted into an acceptor antibody, in order to preserve thebinding region for the TRAIL receptor epitope region. It is appreciatedthat grafting a portion of the total amount of CDRs into a donor isoperative herein. It is understood that grafting generally entails thereplacement, residue for residue, of one amino acid or region, foranother. However, occasionally, especially with the transfer of aregion, one or more residues may be added or omitted or substitutedtherefor, as desired, and that such deletions and insertions, as well asappropriate replacements and inversions, are within the skill of thosein the art.

An antibody of the present invention is obtained by, for example,grafting each CDR of L chain and H chain subunit of an anti-TRAILreceptor monoclonal antibody into a corresponding CDR region of a humanantibody, thereby humanizing a mouse monoclonal antibody effectiveagainst a TRAIL-receptor.

Antibody fragments which contain the idiotype of the molecule are alsogenerated and operative herein using known techniques. For example, suchfragments illustratively include the anti-TRAIL receptor (AB′)2 fragmentwhich can be produced by pepsin digestion of the antibody molecule, theTRAIL receptor antibody AB′ fragments generated through reduction of thedisulfide bridges of the TRAIL receptor (AB′)2 fragment, and theantibody fragment which are generated by treating the antibody moleculewith papain and a reducing agent.

In particular, the anti-DR5 monoclonal antibody TRA-8 may be obtained byculturing a hybridoma which, in turn, may be obtained by immunizing amouse with human DR5 and subsequently fusing the spleen cells or lymphnode cells from the mouse with mouse myeloma cells.

Preparation of a monoclonal antibody illustratively involves thefollowing steps:

-   -   a) purification of a biomacromolecule for use as an antigen;    -   b) preparation of antibody producing cells, after first        immunizing an animal using injections of the antigen, bleeding        the animal and assaying the antibody titer, in order to        determine when to remove the spleen;    -   c) preparation of myeloma cells;    -   d) fusing the antibody producing cells and myeloma cells;    -   e) selecting a hybridoma producing a desired antibody;    -   f) preparing a single cell clone (cloning);    -   g) optionally, culturing the hybridoma cells, or growing animals        into which the hybridoma cells have been transplanted, for large        scale preparation of the monoclonal antibody; and    -   h) testing the biological activities and the specificity, or        assaying marker agent properties, of the monoclonal antibody        thus prepared.

The procedure for the preparation of an anti-DR5 monoclonal antibody isdetailed below with reference to the above-described steps. This methodfor preparing an antibody of the present invention is intended only tobe illustrative of the methods of preparation and is not limitedthereto. Other known procedures may be followed, or the following methodmodified, for instance by using antibody producing cells other thanspleen cells and myeloma.

(a) Preparation of Antigen

A recombinant protein (hereinafter referred to as “recombinant humanDR5”), effective as the antigen, is obtained by transfecting QBI-293Acells with the expression vector pAdDR5-IgG for a fusion proteincomprising the extracellular domain of human DR5 and the Fc region ofhuman IgG1 antibody (hereinafter referred to as “IgG”), (cf. PTA-1428)to express it by using the ADENO-Quest kit (Quantum BiotechnologiesInc., Canada), and collecting and partially purifying the expressionproduct. The plasmid pAdDR5-IgG is constructed by inserting DNA encodinga human DR5 and human IgG fusion protein into pAdCMV5, which is anexpression vector for animal cells. Other materials, such as the DNAencoding DR5, the vector, and the host, are operative herein.

The human DR5 and IgG fusion protein produced in the culture supernatantof the QBI-293A cells transfected with the vector pAdDR5-IgG may bepartially purified by ProteinA-Sepharose affinity chromatography orProteinG-Sepharose affinity chromatography, or ion-exchangechromatography using a Resource Q column (trade name; Pharmacia).

Alternatively, purified DR5 obtained from the cell membranes of humancell lines is used as the antigen. Further, since the primary structuresof DR5 is known (cf. PTA-1428), a peptide comprising the amino acidsequence of SEQ ID NO. 1, may be chemically synthesized by a knownmethod such as the Sanger method, and used as the antigen.

(b) Preparation of Antibody Producing Cells

A mouse is immunized with the immunogen produced in step (a), mixed withan adjuvant, such as Freund's complete or incomplete adjuvant or alum.Other suitable experimental animals illustratively include rats, guineapigs, rabbits, dogs, chickens, horses, pigs, cows and sheep.

Suitable administration routes to immunize an experimental animalinclude the subcutaneous, intraperitoneal, intravenous, intradermal, andintramuscular injection routes, with subcutaneous and intraperitonealinjections being preferred.

Immunizations are optionally performed by a single dose or, by severalrepeated doses at appropriate intervals (preferably 1 to 5 weeks).Immunized animals are monitored for antibody titer in their sera, and ananimal with a sufficiently high antibody titer is selected as the sourceof antibody producing cells. Selecting an animal with a high titer makesthe subsequent process more efficient. Cells for the subsequent fusionare generally harvested from the animal 3 to 5 days after the finalimmunization.

Methods for assaying antibody titer include various well knowntechniques such as radioimmunoassay (hereinafter, referred to as “RIA”),solid-phase enzyme immunoassay (hereinafter, referred to as “ELISA”),fluorescent antibody assay and passive hemagglutination assay, with RIAand ELISA preferred for reasons of detection sensitivity, rapidity,accuracy and potential for automation.

Determination of antibody titer may be performed, for example, by ELISA,as follows. First, purified or partially purified DR5 is adsorbed ontothe surface of a solid phase, such as a 96-well ELISA plate, followed byblocking any remaining surface, to which DR5 has not been bound, with aprotein unrelated to the antigen, such as bovine serum albumin (BSA).After washing, the well surfaces are contacted with serially dilutedsamples of mouse sera to enable binding of the anti-DR5 antibody in thesamples to the antigen. An enzyme-labeled, anti-mouse antibody, as thesecondary antibody, is added to be bound to the mouse antibody. Afterwashing, the enzyme substrate is added, and antibody titer is estimatedby determining absorbance change due to color development caused by thealteration of the substrate or the like.

(c) Preparation of Myeloma Cells

Cells from established mouse cell lines serve as the source of myelomacells, including for example 8-azaguanine resistant mouse, derived fromBALB/c myeloma strains P3X63Ag8U.1 (P3-U1) (35), P3/NS1/1-Ag4-1(NS-1)(36). Sp2/0-Ag14 (SP-2) (37), P3X63Ag8.653 (653) (38) and P3X63Ag8 (X63)(39). The cell line selected is serially transferred into an appropriatemedium, such as 8-azaguanine medium. 8-azaguanine medium includesIscove's Modified Dulbecco's Medium (hereinafter referred to as “IMDM”)or Dulbecco's, Modified Eagle Medium (hereinafter referred to as“DMEM”). RPMI-1640 medium supplemented with glutamine,2-mercaptoethanol, gentamicin, fetal calf scrum (hereinafter referred toas “FCS”), and 8-azaguanine. The cells are then transferred to a normalmedium, such as ASF104 medium (Ajinomoto, K. K.) containing 10% FCS, 3to 4 days prior to fusion, in order to ensure that at least 2×10⁷ cellsare available on the day of fusion.

(d) Cell Fusion

Lymphocytes and plasma cells obtained from any suitable part of theanimal are precursor cells to produce the antibody. Lymphocyte or plasmacell sources illustratively include spleen, lymph nodes, peripheralblood, or any appropriate combination thereof, with spleen cells beingthe most common source.

After the last booster injection, tissue in which antibody producingcells are present is removed from a mouse having the predeterminedantibody titer. The currently favored technique for fusion of spleencells with myeloma cells prepared in step c), employs polyethyleneglycol.

The fusion technique includes washing spleen and myeloma cells withserum-free medium (such as RPMI 1640) or phosphate buffered saline(hereinafter referred to as “PBS”) so that the number ratio of spleencells to myeloma cells is approximately between 5:1 and 10:1, and thencentrifuged. After the supernatant has been discarded and the pelletedcells sufficiently loosened, 1 ml of serum-free medium containing 50%(w/v) polyethylene glycol (m.w. 1,000 to 4,000) is added dropwise withmixing. Subsequently, 10 ml of serum-free medium is slowly added andthen centrifuged. The supernatant is discarded again, and the pelletedcells are suspended in an appropriate amount of HAT medium containing asolution of hypoxanthine, aminopterin and thymidine (hereinafterreferred to as “HAT”) and mouse interleukin-2 (hereinafter referred toas “IL-2”). The suspension is then dispensed into the wells of cultureplates (also referred herein simply as “plates”) and incubated in thepresence of 5% v/v CO₂ at 37° C. for about 2 weeks, with thesupplementary addition of HAT medium as appropriate.

(e) Selection of Hybridomas

When the myeloma strain used is resistant to 8-azaguanine, i.e., it isdeficient in the hypoxanthine guanine phosphoribosyl transferase (HGPRT)enzyme, any unfused myeloma cells and any myeloma-myeloma fusions areunable to survive in HAT medium. On the other hand, fusions of antibodyproducing cells with each other, as well as hybridomas of antibodyproducing cells with myeloma cells can survive, the former only having alimited life. Accordingly, continued incubation in HAT medium results inselection of only the desired hybridomas.

The resulting hybridomas grow into colonies that are then transferredinto HAT medium lacking aminopterin (HT medium). Thereafter, aliquots ofthe culture supernatant are removed to determine anti-Fas antibody titerby, for example, ELISA. When the above-mentioned fusion protein is usedas the ELISA antigen, it is also necessary to eliminate clones producingan antibody which is specifically bound to the Fc region of human IgG1.The presence or absence of such a clone may be verified, for example, byELISA using Fas-IgG1 or IgG1, as the antigen.

(f) Cloning

Hybridomas which have been shown to produce specific antibodies, using amethod similar to that described in step b) to determine antibody titer,are then transferred to another plate for cloning. Suitable cloningmethods include: the limiting dilution method, in which hybridomas arediluted to contain one cell per well of a plate and then cultured; thesoft agar method in which colonies are recovered after culturing in softagar medium; a method of using a micromanipulator to separate a singlecell for culture; and “sort-a-clone,” in which single cells areseparated by a cell sorter.

The cloning procedure according to, for example, the limiting dilutionmethod is repeated 2 to 4 times for each well demonstrating an antibodytiter, and clones having stable antibody titers are selected as anti-DR5monoclonal antibody producing hybridomas. Hybridomas producing an antimouse DR5 antibody are selected by a similar method to obtain ananti-DR5 monoclonal antibody producing cell line.

The mouse-mouse hybridoma TRA-8 which is a basis for antibodies of thepresent invention was deposited with American Type Culture Collection,10801 University Boulevard, Manassas, Va. 20110-2209 in accordance withthe Budapest Treaty on Mar. 1, 2000, and has the accession numberPTA-1428. Accordingly, when preparing an antibody using the mouse-mousehybridoma TRA-8 or any other established hybridoma, the preparation maybe performed by following a procedure starting from the step (g) below,with the steps (a) to (f) omitted.

(g) Culture of Hybridoma to Prepare Monoclonal Antibody

The hybridoma obtained by the cloning is then cultured in normal medium,not in HT medium. Large-scale culture is performed by roller bottleculture, using large culture bottles, or by spinner culture. Thesupernatant from the large-scale culture is then harvested and purifiedby a suitable method, such as gel filtration, which is well known tothose skilled in the art, to obtain an anti-DR5 monoclonal antibodywhich is a basis for antibodies of the present invention. The hybridomamay also be grown intraperitoneally in a syngeneic mouse, such as aBALB/c mouse or a nu/nu mouse, to obtain ascites containing an anti-DR5monoclonal antibody in large quantities. Commercially availablemonoclonal antibody purification kits (for example, MAbTrap GII Kit:Pharmacia) are conveniently used to purify the harvested antibodies.

Monoclonal antibodies prepared as above have a high specificity forhuman DR5.

(h) Assay of Monoclonal Antibody

Suitable identification methods of the isotype and the subclass of themonoclonal antibody include the Ouchterlony method, ELISA and RIA.Preferably, a commercial kit is used for identification, such as a MouseTyper Kit (trade name; BioRad).

Quantification of protein may be performed by the Folin-Lowry method, orby calculation based on the absorbance at 280 nm (1.4(OD280)=Immunoglobulin 1 mg/ml).

Identification of the epitope that the monoclonal antibody recognizes isperformed as follows. First, various partial structures of the moleculethat the monoclonal antibody recognizes are prepared. The partialstructures are prepared by the method wherein various partial peptidesof the molecule are synthetically prepared by known oligopeptidesynthesis technique, or the method wherein DNA encoding the desiredpartial polypeptide is incorporated in a suitable expression plasmid,and is expressed in a suitable host, such as E. coli, to produce thepeptides. Generally, both methods are frequently used in combination forthe above object. For example, a series of polypeptides havingappropriately reduced lengths, working from the C- or N-terminus of theantigen protein, can be prepared by established genetic engineeringtechniques. By establishing which fragments react with the antibody, anapproximate idea of the epitope site is obtained.

The epitope is more closely identified by synthesizing a variety ofsmaller oligopeptides corresponding thereto or mutants of the peptideusing established oligopeptide synthesis techniques to determine abinding property of the peptides to the anti-DR5 monoclonal antibody,for example, which is a basis for preparation of the antibody of thepresent invention and a competitive inhibition of binding of the peptideto an antigen with the monoclonal antibody. Commercially available kits,such as the SPOTs Kit (Genosys Biotechnologies, Inc.) and a series ofmultipin peptide synthesis kits based on the multipin synthesis method(Chiron Corp.) may be conveniently used to obtain a large variety ofoligopeptides.

An antibody of the present invention has the various functionalproperties a) to f) described below, each of which is verified by, forexample, a method described herein below.

a) Specific Binding of TRA-8 to Cells Expressing Human DR5.

A unique feature of the present invention is the ability to bind cellsurface DR5. This is demonstrated by flow cytometry analysis of cellsexpressing DR5. First, specific cell surface binding of DR5 is confirmedby the COS-7 cells transfected with the full-length cDNA encoding humanDR5. Specifically, TRA-8 only recognizes COS-7 cells transfected withDR5 but not empty control vector or vector encoding DR4. Second, threedifferent origins: hematopoietic, glioma, and prostate cancer of humanmalignant tumor cells are tested. The majority of these transformedtumor cells expressed significant levels of cell surface DR5, althoughexpression levels varied largely. Third, two panels of human primarysynovial fibroblast cells from RA and OA patients are examined. All RAsynovial cells expressed significantly higher levels of DR5 compared toOA cells.

b) Induction of Apoptosis of Human Malignant Tumor Cells In Vitro in theAbsence of Crosslinking.

The ability of an antibody raised according to the present invention torecognize TRAIL receptor and to directly induce apoptosis of malignanthuman tumor cells is determined by cell viability assay (ATPLite®)during in vitro culture of cells with various concentrations of anantibody, specifically TRA-8. The majority of tumor cells aresusceptible to TRA-8 induced apoptosis. For some cells, TRA-8 exhibiteda strong apoptosis-inducing activity, for example, TRA-8 is able toinduce apoptosis of human Jurkat cells within the pg/ml levels.Importantly, TRA-8 induced apoptosis did not require crosslinking, andin most cells, TRA-8 exhibited a stronger apoptosis-inducing activitythan the recombinant soluble TRAIL in the presence of the enhancer.

c) Tumoricidal Activity of TRA-8 In Vivo.

Tumoricidal activity of TRA-8 is evaluated in two SCID/human tumor cellmodels. First, SCID mice are intravenously inoculated with humanleukemia Jurkat cells, and treated with a single dose (100 μg) of TRA-8.The results show that the majority of implanted Jurkat cells areeliminated from the peripheral blood and spleen by the treatment withTRA-8, as determined by flow cytometry analysis and in situimmunohistochemical straining of Jurkat cells. Second, human astrocytomacells, 1321N1, are subcutaneously inoculated in SCID mice, and thetumor-bearing mice are treated with a single dose of TRA-8. The growthof implanted 1321N1 cells is significantly inhibited in TRA-8 treatedmice as determined by the sizes of tumor and histological analysis.

d) Identification of RA Synovial Cells by TRA-8

The primary synovial cells isolated from 8 RA and 4 OA patients aretested for cell surface expression of DR5. TRA-8 is able to positivelystrain all RA cells but negatively stain all OA cells. Thus, RA isdifferentiated from OA by the surface expression of DR5 as detected byTRA-8.

e) Induction of Apoptosis in RA Synovial Fibroblast Cells by TRA-8

The ability of TRA-8 to induce apoptosis of RA synovial cells isdetermined by cell viability assay during in vitro culture in thepresence of various concentrations of TRA-8. All RA cells exhibited highto intermediate levels of susceptibility to 100 ng/ml of TRA-8. Incontrast, all OA cells are essentially resistant to TRA-8 inducedapoptosis. Importantly, TRA-8 exhibited a better apoptosis-inducingactivity to RA synovial cells than soluble TRAIL with the enhancer.Moreover, compared to anti-Fas antibody (CH-11). TRA-8 exhibited abetter selectivity to RA synovial cells.

f) TRA-8 does not Induce Production of MMPs in RA Synovial Cells

Since TRA-8 is able to induce NF-kb activation in RA synovial cells asTNF-α, the effect of TRA-8 on the production of MMP1 and MMP3 ofsynovial cells is determined. While TNF-a induced a dose-dependentincrease of MMPs. TRA-8 is unable to induce any production of MMPs, andin some concentrations, TRA-8 slightly decreased the production of MMPsin RA synovial cells.

g) TRA-8 Induces Multiple Caspase Activation.

Since caspases play a crucial role in induction of apoptosis. Theability of TRA-8 to induce caspase activation is determined in humanJurkat cells. When Jurkat cells are incubated with a low dose (50 ng/ml)of TRA-8, the activation of caspase 8, caspase 9, and caspase 3 isobserved as early as 15 minutes after incubation as demonstrated byWestern blot analysis and caspase cleavage analysis. In term of timing,number and strength of caspase activation, antibodies of the presentinvention including the demonstrative antibody TRA-8 exhibited a muchbetter activity than any other known apoptosis-inducing antibodies, suchas anti-human Fas antibody (CH-11).

Thus, an antibody of the present invention is a substance having aproperty to selectively induce apoptosis in pathogenic cells as shown ineffect (a) and (g). Accordingly, it is useful as a prophylactic andtherapeutic agent for diseases associated with inappropriate survival ofcells or inappropriate proliferation of cells, such as thoseattributable to dysregulation of apoptosis systems including the Fas/Fasligand system.

The ability of an antibody of the present invention to induce apoptosisis confirmed by culturing cells such as the human leukemia cell lineJurkat (American Type Culture No. TIB-152) and astrocytoma cell line1321N1 in medium in which the test sample has been added, anddetermining the survival rate by, for example, an ATPLite® assay.

Antibody of the present invention, especially anti-DR5 antibodies havingalmost the same immunogenicity to human as that of human antibodies, isused as an agent for prophylaxis or treatment of diseases associatedwith inappropriate survival or proliferation of cells, including thoseattributable to dysregulation of the apoptosis systems in autoimmunediseases illustratively including systemic lupus erythematosus,Hashimoto's disease, rheumatoid arthritis, graft-versus-host disease,Sjögren's syndrome, pernicious anemia, Addison disease, scleroderma,Goodpasture's syndrome, Crohn's disease, autoimmune hemolytic anemia,sterility, myasthenia gravis, multiple sclerosis, Basedow's disease,thrombopenia purpura, insulin-dependent diabetes mellitus, allergy;asthma, atopic disease; arteriosclerosis; myocarditis; cardiomyopathy;glomerular nephritis; hypoplastic anemia; rejection after organtransplantation and numerous malignancies of lung, prostate, liver,ovary, colon, cervix, lymphatic and breast tissues.

Such a prophylactic or therapeutic agent may be administered in variousforms. Suitable modes of administration include oral administration,such as by tablets, capsules, granules, powders and syrups, orparenteral administration, such as by injection or suppositories.

The antibody or therapeutic agent may be administered orally, rectally,intracisternally, intraventricular, intracranial, intrathecal,intravaginally, parenterally (intravenously, intramuscularly, orsubcutaneously), locally (powders, ointments, or drops), byintraperitoneal injection, transdermally, by inhalation or as a buccalor nasal spray. The exact amount of the antibody or therapeutic agentrequired will vary from subject to subject, depending on the age, weightand general condition of the subject, the severity of the disease thatis being treated, the location and size of the tumor, the particularcompounds used, the mode of administration, and the like. An appropriateamount may be determined by one of ordinary skill in the art using onlyroutine experimentation given the teachings herein. Typical singledosages of antibody range from 0.1-10.000 micrograms, preferably between1 and 100 micrograms. Typical antibody concentrations in a carrier rangefrom 0.2 to 2000 nanograms per delivered milliliter.

Depending on the intended mode of administration, the antibody ortherapeutic agent can be in pharmaceutical compositions in the form ofsolid, semi-solid or liquid dosage forms, such as, for example, tablets,suppositories, pills, capsules, powders, liquids, or suspensions,preferably in unit dosage form suitable for single administration of aprecise dosage. The compositions will include an effective amount of theselected substrate in combination with a pharmaceutically acceptablecarrier and, in addition, may include other medicinal agents,pharmaceutical agents, carriers, or diluents. By “pharmaceuticallyacceptable” is meant a material that is not biologically or otherwiseundesirable, which can be administered to an individual along with theselected substrate without causing significant undesirable biologicaleffects or interacting in a deleterious manner with any of the othercomponents of the pharmaceutical composition in which it is contained.

Compositions suitable for parenteral injection may comprisephysiologically acceptable sterile aqueous or nonaqueous solutions,dispersions, suspensions or emulsions, and sterile powders forreconstitution into sterile injectable solutions or dispersions.Examples of suitable aqueous and nonaqueous carriers, diluents, solventsor vehicles include water, ethanol, polyols (propyleneglycol,polyethyleneglycol, glycerol, and the like), suitable mixtures thereof,vegetable oils (such as olive oil) and injectable organic esters such asethyl oleate. Proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersions and by the use of surfactants.

These compositions may also contain adjuvants such as preserving,wetting, emulsifying, and dispensing agents. Prevention of the action ofmicroorganisms can be ensured by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, sorbic acid, andthe like. It may also be desirable to include isotonic agents, forexample, sugars, sodium chloride, and the like. Prolonged absorption ofthe injectable pharmaceutical form can be brought about by the use ofagents delaying absorption, for example, aluminum monostearate andgelatin.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activecompound is admixed with at least one inert customary excipient (orcarrier) such as sodium citrate or dicalcium phosphate or (a) fillers orextenders, as for example, starches, lactose, sucrose, glucose,mannitol, and silicic acid, (b) binders, as for example,carboxymethylcellulose, alignates, gelatin, polyvinylpyrrolidone,sucrose, and acacia, (c) humectants, as for example, glycerol, (d)disintegrating agents, as for example, agar-agar, calcium carbonate,potato or tapioca starch, alginic acid, certain complex silicates, andsodium carbonate, (e) solution retarders, as for example, paraffin, (f)absorption accelerators, as for example, quaternary ammonium compounds,(g) wetting agents, as for example, cetyl alcohol, and glycerolmonostearate, (h) adsorbents, as for example, kaolin and bentonite, and(i) lubricants, as for example, talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate, or mixturesthereof. In the case of capsules, tablets, and pills, the dosage formsmay also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethyleneglycols, andthe like.

Solid dosage forms such as tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells, such as entericcoatings and others well known in the art. They may contain opacifyingagents, and can also be of such composition that they release the activecompound or compounds in a certain part of the intestinal tract in adelayed manner. Examples of embedding compositions which can be used arepolymeric substances and waxes. The active compounds can also be inmicro-encapsulated form, if appropriate, with one or more of theabove-mentioned excipients.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, and elixirs. Inaddition to the active compounds, the liquid dosage forms may containinert diluents commonly used in the art, such as water or othersolvents, solubilizing agents and emulsifiers, as for example, ethylalcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl alcohol, benzyl benzoate, propyleneglycol,1,3-butyleneglycol, dimethylformamide, oils, in particular, cottonseedoil, groundnut oil, corn germ oil, olive oil, castor oil and sesame oil,glycerol, tetrahydrofurfuryl alcohol, polyethyleneglycols and fatty acidesters of sorbitan or mixtures of these substances, and the like.

Besides such inert diluents, the composition can also include adjuvants,such as wetting agents, emulsifying and suspending agents, sweetening,flavoring, and perfuming agents.

Suspensions, in addition to the active compounds, may contain suspendingagents, as for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, or mixtures of thesesubstances, and the like.

Compositions for rectal administrations are preferably suppositorieswhich can be prepared by mixing the compounds of the present inventionwith suitable non-irritating excipients or carriers such as cocoabutter, polyethyleneglycol or a suppository wax, which are solid atordinary temperatures but liquid at body temperature and therefore, meltin the rectum or vaginal cavity and release the active component.

Dosage forms for topical administration of a compound of this inventioninclude ointments, powders, sprays, and inhalants. The active componentis admixed under sterile conditions with a physiologically acceptablecarrier and any preservatives, buffers, or propellants as may berequired. Ophthalmic formulations, eye ointments, powders, and solutionare also contemplated as being within the scope of this invention.

The term “pharmaceutically acceptable salts, esters, amides, andprodrugs” as used herein refers to those carboxylate salts, amino acidaddition salts, esters, amides, and prodrugs of the compounds of thepresent invention which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of patients without unduetoxicity, irritation, allergic response, and the like, commensurate witha reasonable benefit/risk ratio, and effective for their intended use,as well as the zwitterionic forms, where possible, of the compounds ofthe invention. The term “salts” refers to the relatively non-toxic,inorganic and organic acid addition salts of compounds of the presentinvention. These salts can be prepared in situ during the finalisolation and purification of the compounds or by separately reactingthe purified compound in its free base form with a suitable organic orinorganic acid and isolating the salt thus formed. Representative saltsinclude the hydrobromide, hydrochloride, sulfate, bisulfate, nitrate,acetate, oxalate, valerate, oleate, palmitate, stearate, laurate,borate, benzoate, lactate, phosphate, tosylate, citrate, maleate,fumarate, succinate, tartrate, naphthylate mesylate, glucoheptonate,lactobionate, methane sulphonate and laurylsulphonate salts, and thelike. These may include cations based on the alkali and alkaline earthmetals, such as sodium, lithium, potassium, calcium, magnesium, and thelike, as well as non-toxic ammonium, quaternary ammonium and aminecations including, but not limited to ammonium, tetramethylammonium,tetraethylammonium, methylamine, dimethylamine, trimethylamine,triethylamine, ethylamine, and the like. (See, for example, S. M. Bargeet al., “Pharmaceutical Salts,” J. Pharm. Sci., 1977, 66:1-19 which isincorporated herein by reference.)

The term “prodrug” refers to compounds that are rapidly transformed invivo to yield the parent compounds of the above formula, for example, byhydrolysis in blood. A thorough discussion is provided in T. Higuchi andV. Stella, “Pro-drugs as Novel Delivery Systems,” Vol. 14 of the A.C.S.Symposium Series, and in Bioreversible Carriers in Drug Design, ed.Edward B. Roche, American Pharmaceutical Association and Pergamon Press,1987.

A target cell is a cell of an animal illustratively including human,non-human primate, rat, mouse, guinea pig, rabbit, goat, sheep, cow,horse, chicken, pig, marmoset and ferret.

In addition, the antibody or therapeutic agent of the present inventioncan exist in unsolvated as well as solvated forms with pharmaceuticallyacceptable solvents such as water, ethanol, and the like. In general,the solvated forms are considered equivalent to the unsolvated forms forthe purposes of the present invention.

Antibody molecules are purified by known techniques illustrativelyincluding amino absorption or amino affinity chromatography,chromatographic techniques such as high pressure liquid chromatography,or a combination thereof.

Another aspect of the present invention includes a pharmaceuticalproduct for use in delivering biologically active anti-TRAIL receptorantibody or humanized anti-TRAIL receptor antibody to a vertebrate. Thepharmaceutical product includes a pharmaceutically effective quantity ofanti-TRAIL receptor antibody or fragment thereof, a pharmaceuticallyacceptable carrier, and a container enclosing the carrier and theantibody in a sterile fashion.

In a preferred embodiment of the invention, a pharmaceutically effectiveamount of an anti-DR5 antibody inhibits cell proliferation by contactwith a target cell. A pharmaceutically effective amount of an antibodyrecognizing DR5 or a humanized antibody recognizing DR5 is an amountadministered to an individual sufficient to cause a desired effect.Desired effects of administration of a pharmaceutically effective amountof DR5 recognizing antibodies include death of a target cell, growthinhibition of a target cell, stimulation of DR5, binding to DR5 andincreased NFkB levels or activity in a target cell. A target cell is acell that expresses DR5 and illustratively includes abnormally growingcells and tumor cells such as papillomas and warts; breast cancer, coloncancer, hepatomas, leukemias, lung cancer, melanoma, myelomas,osteosarcomas, ovarian cancer, pancreatic cancer, prostate cancer,cancer of the head and neck, thyroid cancer, uterine cancer and tumorsof the brain such as astrocytomas. In vivo, the target cell is a cell ofan individual with a pathological condition, including those where cellproliferation is abnormal or dysregulated such as malignant or benigncancer and rheumatoid arthritis.

In another preferred embodiment, the target cell is also contacted by atherapeutic agent.

A therapeutic agent is a compound or composition effective inameliorating a pathological condition. An illustrative example of atherapeutic agent includes an anti-cancer compound.

An anti-cancer compound is a compound or composition effective ininhibiting or arresting the growth of an abnormally growing cell. Apharmaceutically effective amount of an anti-cancer compound is anamount administered to an individual sufficient to cause inhibition orarrest of the growth of an abnormally growing cell. Illustrativeexamples of anti-cancer compounds include: bleomycin, carboplatin,chlorambucil, cisplatin, colchicine, cyclophosphamide, daunorubicin,dactinomycin, diethylstilbestrol doxorubicin, etoposide, 5-fluorouracil,floxuridine, melphalan, methotrexate, mitomycin, 6-mercaptopurine,teniposide, 6-thioguanine, vincristine and vinblastine. Further examplesof anti-cancer compounds and therapeutic agents are found in The MerckManual of Diagnosis and Therapy, 15th Ed., Berkow et al., eds., 1987,Rahway, N.J. and Sladek et al. Metabolism and Action of Anti-CancerDrugs, 1987, Powis et al. eds., Taylor and Francis, New York, N.Y.

Antibody of the present invention can be further combined with othertherapies, such as chemotherapy and radiotherapy in the treatment ofmalignance, and therapeutic efficacy can be enhanced byapoptosis-inducing compounds such as bisindolylmaleimide VIII.

Compared to previously published anti-DR5 antibody (24), theapoptosis-inducing activity of the demonstrative TRA-8 antibody of thepresent invention is very strong, and is able to induce apoptosis ofJurkat cells with the pg/ml levels in vitro and demonstrates superior invive tumoricidal activity as compared to previously reported solubleTRAIL. The intravenous administration of a single dose of TRA-8 issufficient to inhibit the growth of both solid tumor and hematopoietictumor cells, whereas induction of in vivo tumor regression with thesoluble TRAIL requires much high dose (500 μg every day for 10 days).The anti-TRAIL receptor antibodies of the present invention appear to beas safe as soluble TRAIL since exemplary antibody TRA-8 does not induceapoptosis of non-transformed fibroblast cells.

Vectors of the present invention include a nucleic acid sequenceencoding a heavy or light chain immunoglobulin of an anti-DR5 antibodyoperably linked to a regulatory element such as a promoter or enhancer.“Operably linked” refers to an arrangement of nucleotide sequencesconfigured so as to perform their usual function. Thus, a regulatoryelement operably linked to a nucleotide sequence encoding a polypeptideis capable of directing the transcription, replication and/ortranslation of the polypeptide. It will be recognized by those skilledin the art that a single vector optionally includes coding sequences forboth a heavy and a light chain immunoglobulin of an anti-DR5 antibody.

The following examples are set forth below to illustrate the methods andresults according to the present invention. These examples are notintended to be inclusive of all aspects of the present invention, butrather to illustrate representative methods and results. These examplesare not intended to exclude equivalents and variations of the presentinvention which are apparent to one skilled in the art.

Example 1. Preparation of DR5 Antigen

1.1 Cloning of DR5 cDNA

DNA encoding the human DR5 protein is cloned by the following RT-PCRmethod using:

a) Template

The total RNA of HeLa cells is extracted by using TRIzol Reagent (GIBCOBRL). The template for the PCR reaction used cDNA that is obtained byusing the First-Strand cDNA synthesis kit (Amersham Pharmacia Biotech)according to the instruction manual provided with the kit.

b) PCR Primers

The following oligonucleotide primers are synthesized for the PCR:

(DR5p1: SEQ ID No. 1) 5′-gacgatgcccgatctactttaaggg-3′;(DR5p2: SEQ ID No. 2) 5′-ccactgggtgatgttggatggg-3′;

Unless otherwise specified, all oligonucleotides in these Examples aresynthesized by Lifetechnologies. All oligonucleotides are stored at −20°C. after being dissolved in distilled water.

c) PCR Reaction

Composition of the PCR reaction solution:

-   -   template cDNA, 5 μl of total 33 μl reaction    -   primer DR5p1, 10 pmol;    -   primer DR5p2, 10 pmol;    -   10× concentrated PCR buffer (provided with the kit), 10 μl;    -   dNTPs (each 2.5 mM), 4 μl; and    -   Taq polymerase (Promega), 5 units.

Sterile distilled water is added to the solution to a total volume of100 μl. Unless otherwise specified, dNTPs are an equimolar mixture ofdATP, dCTP, dGTP and dTTP (2.5 mM each).

The PCR reaction is conducted as follows. The solution is first heatedat 94° C. for 2 minutes, after which a cycle of heating to 94° C. for 30sec, 52° C. for 1 minute and 72° C. for 3 minutes, is repeated 40 times.After completion of this procedure, the reaction solution is heated at72° C. for 10 minutes.

The amplified DNA fragments, thus obtained, are separated on a 1%agarose gel containing 0.25 ug/ml ethidium bromide. The bands determinedto contain the desired DNA fragments are cut out using a razor blade andthe DNA is recovered therefrom using the Gene Clean kit (BOI101). TheDNA fragment is cloned using the TA Cloning Kit (Invitrogen, CA). Thisis performed as follows.

The DNA fragment recovered from the PCR reaction solution, together with50 ng of pCR2.1 vector which is provided with the TA Cloning kit, ismixed with 1 μl of 10× ligase reaction buffer (6 mM Tris-HCl (pH 7.5), 6mM magnesium chloride, 5 mM sodium chloride, 7 mM β-mercaptoethanol, 0.1mM ATP, 2 mM DTT, 1 mM spermidine, and 0.1 mg/ml bovine serum albumin),to which 4 units of T4 DNA ligase (1 μl) has been added. The totalvolume of the mixture is adjusted to 10 μl with sterile deionized water,and the resulting ligase solution is incubated at 14° C. for 15 hours.After this time, 2 μl of the ligase reaction solution is added to 50 μlof competent E. coli strain TOP10F′, which is provided with the TAcloning kit and brought to competence in accordance with the instructionmanual, to which 2 μl of 0.5 M β-mercaptoethanol has been added, and theresulting mixture is kept on ice for 30 minutes, then at 42° C. for 30seconds, and again on ice for 5 minutes. Next, 500 μl of mediumcontaining 2% v/v tryptone, 0.5% w/v yeast extract, 0.05% w/v sodiumchloride, 2.5 mM potassium chloride, 1 mM magnesium chloride, and 20 mMglucose (hereinafter referred to as “SOC” medium) is added to theculture, and the mixture is incubated for 1 hour at 37° C. with shaking.After this time, the culture is spread on an L-broth agar plate (1% v/vtryptone, 0.5% w/v yeast extract, 0.5% w/v sodium chloride, 0.1% w/vglucose, and 0.6% w/v bacto-agar (Difco)), containing 100 μg/ml.Ampicillin resistant colonies appearing on the plate are selected andscraped off with a platinum transfer loop, and cultured in L-brothmedium containing 100 μg/ml ampicillin at 37° C., overnight, withshaking at 200 r.p.m. After incubation, the cells are harvested bycentrifugation, from which plasmid DNA is prepared by the alkali method.EcoRI-EcoRI DR5cDNA fragment from the thus obtained plasmid is subclonedinto pcDNA3 plasmid (Invitrogen, CA). The full length of the DR5 gene inpcDNA3 are sequenced and matched the published sequence. The thusobtained plasmid is designated as plasmid pcDNA3-DR5.

1.2 Construction of DR5-IgG Expression Vector

In order to obtain a soluble form of human DR5 lacking the transmembranedomain, an expression plasmid vector is constructed. This vector isdesigned to encode a fusion protein comprising the extracellular domainof human DR5 fused to the human IgG Fe DNA (41). DNA encoding the humanDR5 lacking the transmembrane domain is obtained by the following PCRreaction.

a) Template

The template for the PCR reaction used pcDNA3-DR5.

b) PCR Primers

The following oligonucleotide primers are synthesized for the PCR:

(DR5p1: SEQ ID No. 1) 5′-gacgatgcccgatctactttaaggg-3′;(DR5p3: SEQ ID No. 3) 5′-ggatccgtggacacattcgatgtc-3′;

Unless otherwise specified, all oligonucleotides in these Examples aresynthesized by Lifetechnologies. All oligonucleotides are stored at −20°C. after being dissolved in distilled water.

c) PCR Reaction

The PCR reaction is conducted and amplified DNA isolated as per Example1.1(c).

The thus obtained plasmid is designated as plasmid pCR-ΔDR5. TheBamHI-EcoRI fragment encoded human Fe fragment which is recovered frompmFas-hIgG1Fc is subcloned into BamHI and EcoRI multi-cloning sites ofpcDNA3. The plasmid thus obtained is designated pcDNAFc. Furthermore,the BamHI-BamHI fragment encoding the human soluble DR5 region which isrecovered from pCR-ΔDR5 is subcloned into the BamHI site of pcDNAFcplasmid. The thus obtained plasmid is designated as plasmidpcDNAΔDR5-Fc. The EcoRI fragment encoding the human soluble DR5-humanIgG Fc region which is recovered from the pcDNAΔDR5-Fc plasmid is bluntended by using the DNA polymerase Klenow fragment (GIBCO BRL) and thensubcloned into the shuttle vector pAdCMV5 (Quantum Biotechnologies Inc.,Canada) which is blunt ended after cutting by BamHI. The plasmid thusobtained is designated pAdΔDR5-Fc.

1.3 Expression and Purification of the Human DR5-IgG1 Fusion Protein

QBI-293A cells (provided with the ADENO-Quest Kit) are co-transfectedwith pAdΔDR5-Fc and QBI-viral DNA (provided with the ADENO-Quest Kit)using the ADENO-Quest kit (Quantum Biotechnologies Inc., Canada)according to the instruction manual. The recombinant virus plaques arecultured and screened for expression of DR5-IgG fusion protein by ELISAanalysis of the supernatant. The positive plaques are amplified inQBI-293A cells and stored at −80° C. as virus stock. Fifty dishes (150mm) of QBI-293A cells are transfected with pAdΔDR5-Fc recombinant virusat 10 m.o.i. (Multiplicity of Infection). The culture media areharvested after transfection for 48 hours.

The transfected cells having the DR5-IgG gene are grown to a celldensity of 1×10⁶ cells/ml by incubation in 500 ml of DMEM (GIBCO)medium, containing 10% v/v FCS, at 37° C. in an atmosphere of 5% v/v CO₂for 2 days. The culture is then centrifuged (1,000 r.p.m., 5 minutes)and the supernatant collected. The purification of DR5-IgG from thesupernatant is achieved using ProteinA-Sepharose CL-4B affinitychromatography (Pharmacia) under the following conditions:

-   -   column: ProteinA-Sepharose CL-4B column (column size 2 ml;        Pharmacia);    -   elution buffer: 0.1 M glycine (pH 2.4), 0.15 M NaCl;    -   neutralization buffer: 1M Tris-HCl (pH 8.5).

After all of the supernatant is applied to the column, it is washedthree times with 20 ml of PBS and then 1 ml of elution buffer is added10 times. The optical density of each eluted fraction (1 ml) ismeasured. The second fraction through the fifth fraction (withOD₂₈₀≧0.1) are collected and after addition of 100 μl of neutralizationbuffer, the eluates are placed separately in dialysis tubing, and theeluates dialyzed against 1 liter of PBS (pH 7.5) at 4° C. The dialysisbuffer being changed twice.

The eluates are then assayed for expression of the DR5-IgG gene productby ELISA. First, 100 μl of each fraction are placed separately intowells of a 96-well microplate (Costar) and incubated at 37° C. for 1hour. After this time, the solution in the wells is removed, and theplate is washed 3 times with 100 μl/well of PBS containing 0.1% v/vTween® 20 (hereinafter referred to as “PBS-Tween”). After washing, PBScontaining 2% w/v bovine serum albumin (hereinafter referred to as“BSA”) is added in quantities of 100 μl/well, and the plate is thenincubated at 37° C. for 1 hour. After this time, the wells are washed afurther 3 times with 100 μl/well of PBS-Tween, after which 100 μl/wellof a solution of anti-human IgG1 monoclonal antibody diluted 1000-foldwith PBS-Tween is added to each well, and the plate is once againincubated at 37° C. for 1 hour. The wells are then washed 3 times with100 μl/well of PBS-Tween. 3,3′,5,5′-Tetramethyl-benzidine (hereinafterreferred to as “TMB”) liquid substrate system (Sigma) is then added inan amount of 100 μL/well and the plate is allowed to stand at roomtemperature for 5 minutes and then the reaction stopped by adding 100μl/well of 0.2N H₂SO₄. The absorbance of each well is read at 450 nm toestimate the concentration of the bound antibody, using the absorbanceat 650 nm as the control reading. The absorbance is measured using amicroplate reader (Molecular Devices). The production of DR5-IgG1 isconfirmed using this ELISA method. The molecular weight of the expressedDR5-IgG1 fusion protein is determined using western blotting analysis inwhich anti-human IgG1 mAb (Sigma) is used to detect the antibody on thegel. The molecular weight of the expressed DR5-IgG1 fusion protein hasan approximate molecular weight of 50 kDa. The purity achieved beinggreater than 90% as evaluated by analysis on SDS-PAGE and detection ofthe protein by Coomassie blue staining.

Example 2. Generation of Monoclonal Antibodies Against Human DR5

2.1 Immunization

Female, Balb/c mice (Jackson Laboratory, Bar Harbor, Me.) of 6-8 weeksof age, are immunized with the affinity-purified human DR5/hIgG1 fusionprotein. For the initial foot-pad immunization, the fusion protein (50μg) is emulsified in Freund's complete adjuvant (Difco, Detroit, Mich.).The mice are then boosted with four injections of 50 μg of fusionprotein administered without adjuvant every other day. Three days afterthe last injection, lymphocytes from the local lymph nodes are fusedwith NS-1 myeloma cells, and the hybridomas are cultured in F104 mediasupplemented with 10% fetal calf serum. Positive hybridomas are selectedby ELISA in which the plates are coated either with 1 μg/ml DR5/hIgG1 orthe same amount of Fas/hIgG1 as a control. The isotype of the hybridomasis determined by ELISA using a panel of mouse Ig isotype-specific goatantibodies (Southern Biotechnology, Birmingham, Ala.). Monoclonalantibodies are purified by affinity chromatography using immobilizedanti-mouse IgG1 or protein G (Sigma).

2.2 Cell Fusion

On the third day after the booster injection, the local lymph nodes areremoved from the mouse and placed into 10 ml of serum-free RPMI 1640medium (GIBCO BRL) containing 50 units/ml penicillin, 50 μg/mlstreptomycin, and 300 μg/ml L-glutamic acid, and disrupted by passingthe organ through a mesh (Cell Strainer; Falcon) using a spatula. Theresulting cell suspension is centrifuged to pellet the local lymph nodescells which are then washed twice with serum-free RPMI medium. Thewashed cells are then resuspended in scrum-free RPMI medium and counted.

In the meantime, myeloma NS1 cells (American Type Culture CollectionTIB-18) had been grown to a cell density not exceeding 1×10 cells/ml inASF104 medium (Ajinomoto, K. K.) containing 10% v/v FCS (Gibco BRL)(“ASF medium with serum”) at 37° C. under 5% v/v CO₂, and these arelikewise disrupted, washed, resuspended and counted.

An amount of the NS1 cell suspension calculated to contain 3×10⁷ cellsis mixed with an amount of the spleen cell suspension calculated tocontain 3×10⁸ cells. The resulting mix is centrifuged and thesupernatant discarded. The following steps of the cell fusion areperformed whilst at all times keeping the plastic tube containing thepellet at 37° C. in a beaker of warm water.

One ml of 50% (w/v) polyethylene glycol 1500 (Boehringer Manheim) isthen slowly added to the tube, all the while stirring the pellet usingthe tip of a pipette. Subsequently, 1 ml of serum-free RPMI medium,prewarmed to 37° C., is slowly added in 2 portions, followed by theaddition of a further 7 ml of serum-free RPMI medium. The resulting mixis then centrifuged, the supernatant discarded and 10 ml of HAT mediumcontaining 10% v/v FCS are added while stirring gently with the tip of apipette. A further 20 ml of HAT medium containing 10% v/v FCS is added,and the suspension is dispensed into 96-well cell culture microplates at100 μl/well and incubated at 37° C. in an atmosphere of 5% v/v CO₂.After 7 or 8 days, 100 μl/well of fresh HAT medium are used to replacemedium in any wells exhibiting a yellowish hue. The fusion cells fromthese wells are cloned by limiting dilution as described below.

2.3 Cloning by Limiting Dilution

Thymuses from 4 to 10 week-old female BALB/c mice (from Japan SLC, Inc.)are removed, disrupted on a mesh (Cell Strainer; Falcon) as describedabove, and the disrupted cells are washed twice with HT mediumcontaining 10% v/v FCS. An amount of thymus cells corresponding to thosefrom one mouse is suspended in 30 ml of HT medium containing 10% v/v FCSto produce a feeder cell suspension. The fusion cell preparationobtained above in Example 2.2 is diluted with this feeder cellsuspension 10- to 100-fold, and further diluted serially with the feedercell suspension to make suspensions having fusion cell densities of 5, 1and 0.5 cells/ml. The thus prepared samples are dispensed into wells of96-well cell culture microplates at 100 μL/well and incubated for 5 daysat 37° C. under 5% v/v CO₂.

2.4 Screening

The culture supernatants from the growing hybridomas are screened byELISA using plates coated either with 1 μg/ml DR5/hIgG1 or the sameamount of Fas/IgG1 (41) as a control. The bound antibodies are detectedusing horseradish peroxidase (HRP)-conjugated anti-mouse immunoglobulins(Southern Biotechnology. Birmingham, Ala.) with TMB (Sigma, St Louis,Mich.) as the substrate. Purified DR5-IgG1 at a concentration of 1 μg/mlor the same amount of Fas-hIgG1 are introduced into a well of a 96-wellELISA/RIA STRIP PLATE (Costar, N.Y.). The plate is kept standing at 4°C. overnight to allow adsorption of the protein onto the well surface.After this time, the solution in the wells is discarded and each well iswashed 3 times with PBS-Tween. Then, 100 μl of PBS containing 1% (w/v)bovine serum albumin (A3803; Sigma Chemicals Co.) is added to each welland the plate is incubated at 37° C. for 1 hour. The wells are thenwashed a further 3 times with PBS-Tween, and then 50 μl of each culturesupernatants from the growing hybridomas is added to each well. Theplate is then incubated at 37° C. for 1 hour, and the wells are againwashed 4 times with PBS-Tween. After washing, 50 μl of horseradishperoxidase labeled goat anti-mouse immunoglobulin antibody (SouthernBiotechnology. Birmingham, Ala.), diluted 1000-fold with PBS, is addedper well, and the plate is again incubated at 37° C. for 1 hour, afterwhich the wells are washed 4 times with PBS-Tween.3,3′,5,5′-Tetramethyl-benzidine (TMB) liquid substrate system (Sigma) isthen added in an amount of 100 μl/well and the plate is allowed to standat room temperature for 5 minutes and then the reaction stopped byaddition of 100 μl/well of 0.2N H₂SO₄. The absorbance of each well at450 nm (control 650 nm) is measured using a microplate reader (MolecularDevices) and fusion cells are selected from the sample which had theabsorbance (450 nm-650 nm, OD values; >0.5) clearly higher than those towhich no fusion cells supernatant had been added (OD values; ≈0.03).

Furthermore, the culture supernatants from the growing hybridomas arealso functionally screened by measuring the apoptosis-inducing activityusing Jurkat cell. Fifty μl of RPMI medium containing Jurkat cells (1000cells per well) and 5 uM Bisindolylmaleimide VIII (BisVIII, Alexis, SanDiego, Calif.) are added in 96-well plates in the presence of 50 μl ofthe culture supernatants from the growing hybridomas. The cells arecultured in a humidified incubator at 37° C.: overnight. Apoptosis isdetermined by cell viability using the ATPLite® kit as instructed by themanufacturer (Packard Instruments), and the samples are counted usingthe TopCounter (Packard Instruments).

2.5 ELISA Binding of TRAIL and TRA-8 to the Receptors

ELISA plates are coated with 2 μg/ml of DR4-Ig or DR5-Ig fusion proteinovernight. After blocking with 3% BSA, the soluble TRAIL-FLAG or TRA-8is added at indicated concentrations and incubated at 37° C. for onehour. The binding of TRAIL or TRA-8 is detected by HRP-conjugatedanti-Flag antibody (Alexis) or HRP-conjugated anti-murine IgG1 (SouthernBiotechnology), respectively. The reactions are developed by TMBsubstrate buffer and measured by the Benchmark Microplate Reader(BioRad). The Kd values are estimated by the one-site binding model ofnon-linear regression using GraphPad Prism® software (GraphPad Software,San Diego, Calif.). For competitive ELISA, 100 ng/ml TRAIL-FLAG is addedand incubated in the presence of various concentrations of TRA-8. Thebinding of TRAIL is determined as above.

2.6 Cloning

The steps described in Examples 2.3 and 2.4 above are repeated 5 timesfor the cells selected in 2.4, thereby enabling the selection of severalhybridoma clones each of which produced a single antibody that boundDR5-IgG but did not bind Fas-IgG. As a result of this selectionprocedure, a mouse-mouse hybridoma, designated TRA-8 and producing anantibody binding to DR5-IgG but not Fas-IgG, is obtained. Thishybridoma, TRA-8, was deposited with the American Type CultureCollection on Mar. 1, 2000, and has been assigned accession No.PTA-1428.

The subclass of the antibody produced by the mouse-mouse hybridoma TRA-8(hereinafter referred to simply as “TRA-8”) is demonstrated to be IgG1,κ, after testing with a monoclonal antibody isotyping kit (Pierce).

Using our human DR5-IgG1 fusion protein as immunogen, seven hybridomaclones are obtained by initial ELISA screening, all of which arestrongly positive for DR5-IgG but not the Fas-IgG fusion protein,indicating that the obtained hybridomas produce antibodies thatrecognize the extracellular portion of DR5 but not the Fe portion ofIgG1 (data not shown).

2.7 Western Blot Analysis

Filters for Western blot analysis of normal human and cancer tissuehomogenates are purchased from Geno Technology (St Louis, Mo.). Eachlane is loaded with an equal amount of protein as determined by ananti-β-actin antibody. The blots are probed with 1 μg/ml TRA-8overnight, and followed by HRP-conjugated goat anti-mouse IgG1 (SouthernBiotechnology) at room temperature for one hour, and developed bychemiluminescence.

2.8 In Situ Immunohistochemistry

Human tissues are obtained from the Tissue Procurement Center of UAB.Frozen sections are fixed in 70% ethanol, blocked with 10% horse serumin PBS, and then incubated with 10 μg/ml of affinity-purified TRA-8 atroom temperature for 60 minutes. The anti-mouse IgG ABC kit withdiaminobenzidine (Vector, Burlingame, Calif.) as the colorimetricsubstrate is used to visualize the reactivity.

2.9 Analysis of Caspase Activation

Jurkat cells (1×10⁶/ml) are incubated with 500 ng/ml TRA-8. Aliquots (30μg of protein) of the cell lysate are separated on 15% SDS-PAGE, blottedonto a nylon membrane, and the blots are probed with anti-caspase 8, 9,and 3 antibodies (BD Pharmingen, San Diego, Calif.) followed byHRP-conjugated secondary antibody and chemiluminescence visualization ofcleaved products. The caspase inhibitor set is purchased from R&DSystems (Minneapolis, Minn.). Each caspase inhibitor is added intoculture at indicated concentrations.

Example 3. Purification of TRA-8 Monoclonal Antibody

The mouse-mouse hybridoma, TRA-8, is grown to a cell density of 1×10⁶cells/ml by incubation in 500 ml of ASF medium, containing 10% v/v FCS,at 37° C. under 5% v/v CO₂ for 5 days. The culture is then centrifuged(1,000 r.p.m., 5 minutes) and the supernatant collected. Thepurification of TRA-8 from the supernatant is achieved usingProteinG-Sepharose CL-4B affinity chromatography (Pharmacia) under thefollowing conditions:

-   -   column: ProteinG-Sepharose CL-4B column (column size 2 ml;        Pharmacia);    -   elution buffer: 0.1 M Glycine (pH 2.4), 0.15 M NaCl;    -   neutralization buffer: 1M Tris-HCl (pH 8.5).

After all of the supernatant is applied to column, 20 ml of PBS iswashed three times and then elution buffer is added in 1 ml volumes for10 times. The optical density of each eluted fraction (1 ml) ismeasured. The fractions from No. 2 to No. 5 (>OD₂₈₀=0.1) are collectedseparately.

After adding 100 μl of neutralization buffer, the eluates are placed indialysis tubing separately, and the eluates dialyzed against 1 liter ofPBS (pH 7.5) at 4° C. The dialysis buffer being changed twice. Thissample is assayed for anti-DR5 antibody activity by ELISA using thehuman DR5-IgG fusion protein prepared above using the techniquedescribed above.

Example 4. Preparation of DR4 Antigen, DR4-IgG Expression Vector andAnti-DR4 Monoclonal Antibody

The procedures of Examples 1-3 are repeated with DR4 template cDNA andprimers in place of those detailed in Example 1 to obtain a DR4 antigenwhich is utilized as per Examples 1.2-3 to obtain a monoclonal antibodyspecific against DR4.

Example 5. Monoclonal Antibodies Against DcR1 and DcR2

Monoclonal antibodies are raised against decoy receptors DcR1 and DcR2by substituting the corresponding cDNA and primers to create therespective antigens as per Example 1. Expression vectors for DcR1 orDcR2-fusions with immune globulin G and resulting purified monoclonalantibodies are created as per Examples 2 and 3.

Example 6. The Specificity of a Monoclonal Antibody

As all of the receptors for TRAIL and other proteins of the TNFR familyshare significant homology, the specificity of exemplary antibody TRA-8for DR5 is determined by western blot analysis using two different humanDR5-IgG fusion proteins and soluble, recombinant forms of other relatedproteins. A first DR5-Ig fusion protein is constructed by fusing cDNAfrom residues 1-180 of the extracellular portion of DR5 and cDNAencoding the constant region of human IgG1. The fused cDNA is clonedinto a recombinant adenoviral vector (Quantum Biotechnogies, Inc.,Montreal, Canada). The expressed DR5/hIgG1 fusion protein, which had arelative molecular weight of 50 kDa, is purified using an anti-human IgGaffinity column (Sigma, St Louis, Mo.). For western blot analysis ofspecificity, a second recombinant human DR5/IgG1 fusion protein (aa.52-212), as well as TRAIL-R1, R3 and R4 fusion proteins, are purchasedfrom Alexis. The soluble forms of human Fas and TNFR1 are kindlyprovided by Dr. Carl Edwards of Amgen, Inc., Thousands Oaks, Calif.,USA. The soluble recombinant human DR4, DcR1, DcR2, TNFR1, R4, and Fasmolecules used are human IgG1 fusion proteins. 0.5 μg of each protein isseparated by 10% SDS-PAGE and blotted onto a nitrocellulose membrane.The blots are blocked with 5% dry milk in PBS at room temperature forone hour, and probed with 1 g/ml of purified monoclonal anti-DR5antibody (clone: TRA-8) or 0.1 μg/ml of HRP-conjugated goat anti-humanIgG at 4° C. overnight. Horseradish-peroxidase (HRP)-conjugated goatanti-mouse IgG is used as secondary antibody to detect bound TRA-8. Theblots are developed by chemiluminescence.

Cos-7 cells transfected with the pcDNA3 vector (Clontech, Palo Alto,Calif.) containing the full-length DR5 or DR4 or empty vector are usedfor flow cytometry analysis. The full-length cDNA encoding human TRAILor murine Fas ligand is cloned into the pTRE vector down-stream of thetetracycline-controllable promoter (Clontech). The XhoI-HindIIIfragments of pTRE-hTRAIL or pTRE-mFasLt are further cloned into theadenoviral shuttle vector pAdBN (Quantum Biotechnologies, Inc.). The 293host cells are co-transfected with the linearized pAd-TRE-hTRAIL orpAd-TRE-mFasL and the large fragment DNA of adenovirus. The expressionof functional human TRAIL or murine Fas ligand from the recombinantvirus plaques is screened using a ⁵¹Cr-release assay with Jurkat as thetargets.

TRA-8 reacted strongly with the DR5-IgG fusion protein (˜50 kDa), whichis used for immunization as shown in FIG. 1a , DR5, #1, and weakly withthe second DR5-IgG fusion protein (˜60 kD) as shown in FIG. 1a , DR5,#2. There is no significant binding of TRA-8 to DR4, DcR1, DcR2, Fas(CD95) or TNFR1. These results indicate that TRA-8 recognizes theepitopes that are specific for DR5 but not shared by the other membersof the family.

TRA-8 does not react with other members of the TNF receptor superfamily,such as Fas (CD95) and TNF receptor 1, nor does TRA-8 cross-react withthe murine homologue of DR5 as shown by optical absorbance ratios for450 nm and 650 nm, wherein lower panel numbers 1-7 (FIG. 1a , the column8 of lower panel). Soluble TRAIL and TRA-8 bound comparably toimmobilized DR5 (FIG. 1b , left panel). In contrast, TRAIL bound to DR4,but TRA-8 did not exhibit any binding activity to DR4 (FIG. 1b , middlepanel). The Kd values for the binding of TRAIL and TRA-8 to DR5 areestimated at 59 nM and 3 nM, respectively. Importantly, TRA-8efficiently competing with TRAIL for binding to DR5 but not for bindingto DR4, as shown in competitive ELISA (FIG. 1b , right panel). Theseresults establish the specificity of TRA-8 for human DR5.

TRA-8 is able to detect cell surface expression of DR5, with flowcytometric analysis indicating specific binding to the cell surface ofCos-7 cells transfected with full-length human DR5, but not of Cos-7cells transfected with DR4 or empty vector (FIG. 1c ). Similarly, insitu immunohistochemistry with TRA-8 demonstrated reactivity with Cos-7cells transfected with full-length DR5 DNA but not with thosetransfected with control vector (FIG. 1d ). TRA-8 does not induceapoptosis of untransfected Cos-7 cells, and RT-PCR of RNA from Cos-7cells using paired primers encoding human DR5 showed that no specificPCR products. Further functional analysis using human Jurkat cells astargets showed that, in the absence of crosslinking, TRA-8 stronglyinduces cell death, demonstrated by three different assays for cellviability including ATPLite®, MTT and P1 exclusion (FIG. 1e ). Greaterthan 50% of Jurkat cells are killed by nanogram levels of TRA-8 as shownby ATPLite® assay. The killing activity of TRA-8 is specific for DR5 asit could be blocked by DR5-Ig but not DR4-Ig fusion protein (data notshown). Cleavage of caspases 8, 9, and 3 could be detected by westernblot analysis as early as 30 minutes after TRA-8 treatment of Jurkatcells (FIG. 1f ), and cell death of Jurkat cells is completely inhibitedby the general caspase inhibitor (Z-VAD) (FIG. 1g ). Individual caspaseinhibitors for caspase 8, 3, 9, and 10 partially inhibited cell death,further indicating that TRA-8-mediated cell death is primarily through acaspase-dependent apoptotic mechanism.

Example 7. Flow Cytometric Analysis of the Expression of Cell SurfaceDR5: A Major Death Receptor on Many Tumor Cells but not on Normal Cells

The ability of TRA-8 to bind DR5 expressed on the cell surface and thespecificity of this reaction is then assessed using COS-7 (American TypeCulture Collection No. CRL-1651) cells transfected with the expressionvector containing the full-length human DR5 or DR4 cDNA or empty vectoras control. Phycoerythrin (PE)-conjugated anti-mouse IgG1 (Pharmingen)is used as the second antibody to detect the bound TRA-8. Thefluorescence of 1×10⁴ cells is measured, using a flow cytometer(FACSVantage™) under the following conditions:

-   -   Excitation wave length: 488 nm;    -   Detection wave length; 600 nm.

Flow cytometry analysis showed that TRA-8 stained approximately 30% ofCOS-7 cells transfected with the DR5 vector as shown in the solidhistogram of FIG. 1c . This percentage parallels the transfectionefficiency as determined by analysis of transfection using greenfluorescent protein (GFP) (data not shown). TRA-8 did not significantlystain cells transfected with either DR4 (the open histogram) or controlvector (the dotted histogram), indicating that TRA-8 is specific forcell surface DR5.

Although DR5 expression in tumor cells has been studied extensively atthe mRNA level (Rieger J. et al. 1:FEBS Lett 1998 May 1; 427(1):124-8),the surface expression of DR5 is not well understood. Gibson S. B. etal. 1:Mol Cell Biol 2000 January; 20(1):205-12; Kim K. et al. 1: ClinCancer Res 2000 February; 6(2):334-46. Thus, the availability ofmonoclonal anti-DR5 antibody allows us to examine the surface levels ofDR5, and to correlate the expression with the susceptibility of thecells to TRAIL-mediated apoptosis. The following panel of cells (1×10⁶)is incubated with 10 μg/ml of affinity purified TRA-8 at roomtemperature for 30 min, and then stained with PE-conjugated anti-mouseIgG1 (Pharmingen) for another 30 min. 10,000 viable cells are analyzedusing the FACS Vantage™ flow cytometer under the following conditions:

-   -   Excitation wave length: 488 nm;    -   Detection wave length: 600 nm.

The five hematopoietic cell lines tested are Jurkat, CEM-6, Molt-4, H-9and U937 cells. DR5 expression is detectable on the surface of Jurkat.CEM-6, H-9, and U937 cells but is almost undetectable on Molt-4 cells asshown in FIGS. 2a and 2a ′. Although high levels of DR5 RNA expressionhas been described previously (43), the FACs analysis indicated thatthese cells do not express high levels of the surface DR5. These resultsindicate that cell surface expression of DR5 does not correlate with thetranscriptional expression of DR5, which is not unexpected for such areceptor. The level of cell surface expression of DR5 may be celllineage-specific since most of the cells of hematopoietic originexpressed low levels whereas most glioma and prostate cells expressedhigh levels of DR5.

TRA-8 monoclonal antibody is used determine the role of DR5 in inductionof TRAIL-mediated apoptosis by examining its cell surface expressionamong a panel of different types of human tumor cells as well as thesusceptibility of these cells to both TRAIL and TRA-8-mediatedapoptosis. Primary peripheral blood T cells did not express significantlevels of cell surface DR5 and are resistant to both TRAIL andTRA-8-mediated apoptosis (FIGS. 2a, 2a ′ and 3 a′). Although all five ofthe human T-leukemia cell lines tested expressed detectable albeitrelatively low levels of cell surface DR5, two of them (Jurkat andCEM-6) are highly susceptible to both TRAIL-mediated and TRA-8-mediatedapoptosis, indicating that DR5 alone is sufficient to induce apoptosisof these cells. Molt-4 and U937 cells are partially susceptible toTRAIL-mediated apoptosis but are relatively resistant to TRA-8-mediatedapoptosis, suggesting that other TRAIL receptors might be involved intransduction of an apoptosis signal. H-9 cells are resistant to bothTRAIL and TRA-8-mediated apoptosis, implicating a block mediated by anintracellular anti-apoptosis pathway.

The panel of cells included the human malignant glioma cell lines,Hs683, U251MG, D37MG, D54MG, U373MG, CH235MG, U87 and normal humanastrocytes, which were provided by Dr. Yancey Gillespie of theNeurosurgery Department of the University of Alabama at Birmingham. Thehuman prostate cancer cell lines, Du154, PC3 and LnCap, were provided byDr. William Grizzle of the Pathology Department of the University ofAlabama at Birmingham who had obtained the cell lines from the AmericanType Culture Collection. The human leukemia 1 cell lines, B-celllymphoma, HepG2 Jurkat (American Type Culture Collection TIB-152) andCCRF-CEM CEM-6 (American Type Culture Collection CCL-119); monocyte celllines, U937 (American Type Culture Collection CRL-2367); were purchasedfrom the American Type Culture Collection. All above cell lines arecultured in RPM 1640 supplemented with 10% FCS. The human astrocytomacell line, 1321N1, was kindly provided by Dr. Richard Jope of PsychiatryDepartment of the University of Alabama at Birmingham, and cultured inDMEM supplemented with 5% FCS.

Soluble recombinant human TRAIL, purchased from Alexis Corporation (SanDiego, Calif.), is a fusion protein comprised of the extracellulardomain of human TRAIL (aa residues 95 to 281) fused at N-terminus to aFLAG-tag and an 8 amino acid linker peptide. Unlike previously reportedHis-tagged TRAIL, this preparation of TRAIL alone does not induce astrong apoptotic response in Jurkat cells and requires an anti-FLAGantibody as a crosslinker to enhance apoptosis. The anti-FLAG antibodywas also purchased from Alexis.

All of the 10 human malignant glioma cells tested expressed detectablelevels of DR5 at the cell surface. Most expressed intermediate to highlevels of DR5 as shown in FIG. 2b . Three lines, D-54MG, U373MG andCH-235MG expressed high levels of DR5 while six lines. Hs-683, U251-MG,D37-MG, U87, SMK1 and 1321N1, expressed intermediate levels of DR5. Onlyone cell line, H-465 expressed low levels of DR5. All three prostatecancer cell lines expressed high levels of DR5 as shown in FIG. 2 c.

Like the normal primary T cells, primary B cells did not expresssignificant levels of DR5 and did not undergo apoptosis after treatmentwith either TRAIL or TRA-8 (FIG. 2d ). Three (SKW6.4, EB-3, and Raji)out of the four B lymphoma cell lines tested expressed relatively highlevels of DR5 and are very susceptible to both TRAIL and TRA-8-mediatedapoptosis. The fourth cell line, Daudi, expressed very low levels of DR5and is much less susceptible to either TRAIL or TRA-8-mediatedapoptosis. Although primary astrocytes did not express detectable levelsof cell surface DR5 (FIG. 2b ′), all four glioma cell lines testedexpressed high levels of DR5. The higher level of expression of DR5 onglioma cells than on T and B cells is not accompanied by a significantlygreater susceptibility to TRAIL and DR5-mediated apoptosis, suggestingthat the level of cell surface expression of DR5 is not necessarilycorrelated with the level of apoptosis of tumor cells. RT-PCR, performedto determine message levels of DR4, DR5 and DCR2, detected message inall cells tested (Table 1). However, in general, primary normal cellsexpressed relatively low levels of DR5 compared to transformed tumorcells.

TABLE 1 RT-PCR analysis of TRAIL receptor expression* Cells DR5 DR4 DcR2Primary T cells <0.001 <0.001 0.015 Jurkat 0.10 <0.001 0.21 CEM-6 0.500.59 0.25 Molt-4 0.10 <0.001 0.05 H-9 0.73 0.61 0.07 Primary B cells<0.001 <0.001 0.024 SKW6.4 0.95 0.66 0.45 EB3 0.40 <0.001 0.35 Raji 0.550.11 0.45 Daudi 0.73 0.36 0.63 Normal Astrocytes 0.05 <0.001 0.12 SH6830.56 0.96 0.14 U87 0.44 0.56 0.21 D54 1.15 0.46 0.12 1321N1 0.25 0.350.05 *Total RNA was isolated from cells and RT-PCR was performed asdescribed in Methods.The PCR products were separated in 3% agarose gel and analyzed by theFluor-S MAX MultiImager System (BioRad). The values are presented as aratio relative to β-actin.

Example 8. Induction of Apoptosis In Vitro in Malignant Cells

To determine whether TRA-8 induces apoptosis in transformed cells invitro, all DR5-positive tumor cells are examined for theirsusceptibility to apoptosis induced either by TRA-8 or TRAIL.

Target cells (1×10³ per well) are cultured in 96-well plates in thepresence of the indicated concentrations of soluble TRAIL pluscrosslinker (Alexis) or TRA-8 at 37° C. overnight. Cell viability isdetermined using (1) the ATPLite® kit according to the manufacturer'sinstructions (Packard Instruments, Meriden, Conn.); (2) the MTT Cellproliferation/viability kit (Sigma); or (3) PI staining of dead cellsand analyzed by flow cytometry. At end of culture, cells are stainedwith 10 μg/ml PI and PI negative cells are gated as viable cells. Foranalysis of condensed nuclei of hepatocytes, cells are stained with 10ng/ml Hoechst 33352 (Molecular Probes) and analyzed by flow cytometry.

The TRA-8 antibody is capable of inducing apoptosis in the majority ofthe malignant human glioma cell lines (9/10), in 2 of the 3 prostatecancer cell lines, and in 2 of the 4 DR5-positive hematopoietic celllines. It did not induce apoptosis in the Molt-4 cell line, whichexpressed almost undetectable cell surface levels of DR5. The levels ofsusceptibility of the cells to TRA-8-mediated apoptosis variedconsiderably among the cell lines, however.

The variability of the susceptibility of the cells to TRA-8 antibodyinduced apoptosis suggests that although a minimal level of cell surfaceexpression of DR5 is required, the level of cell surface expression ofDR5 is not necessarily the primary determinant of susceptibility andother factors influence this process. Although all of the glioma cellsgenerally expressed significantly higher levels of the surface DR5 thandid the hematopoietic cells, glioma cell susceptibility to apoptosisinduced by TRA-8 is not proportionally increased compared to thehematopoietic cells. The susceptibility of five of the glioma celllines, D-37MG, D54-MG, U373-MG, CH235-MG and 1321N1 to TRA-8-inducedapoptosis is high and is equivalent to their susceptibility toTRAIL-mediated apoptosis as shown in FIG. 3b . Two of the glioma celllines, H-456 and SMK1, are much less susceptible to apoptosis induced byTRA-8. In the case of the H-456 cells, the surface expression of DR5 islow; however, the surface expression of DR5 on SMK1 is similar to themore susceptible cell lines, suggesting that other mechanisms might playa role in the determining the susceptibility to TRAIL-mediatedapoptosis. Although all three prostate cancer cell lines expressed highlevels of DR5, the Du145 cells are most sensitive to TRA-8-inducedapoptosis, the PC3 cells are partially sensitive while LnCAP cells arecompletely resistant as shown in FIG. 3c . Among the hematopoieticcells, it is found that Jurkat and CEM-6 are very susceptible toTRA-8-apoptosis as shown in FIG. 2a although both these cell lines hadbeen found to express low levels of DR5. Although DR5 is detectable onU937 cells, these cells are resistant to TRA-8-induced apoptosis.Similarly, although the H-9 cells expressed detectable levels of DR5,H-9 cells are resistant to apoptosis induced by TRA-8. These resultsimplicated the existence of regulatory mechanisms that influenceDR5-mediated apoptosis.

Additional surface binding anti-DR5 antibodies are produced as per theprocedures of Examples 1-3. Two additional anti-DR5 antibodiesdesignated TRA-1 and TRA-10 are studied along with TRA-8 to determinecomparative ability induce apoptosis and thereby act as an agonist orconversely block TRAIL-mediated apoptosis, thereby acting as anantagonist. Human Jurkat cells are used as a target to determine theagonist and/or antagonist activity of the three anti-DR5 antibodiesdenoted TRA-1, TRA-8 and TRA-10. As shown in FIG. 4, cell viability isabout 90%, 70% and 20% for TRA-10, TRA-1 and TRA-8, respectively uponovernight incubation with 2.5 μg per ml. TRA-8 induced a strongapoptotic response in a dose dependent fashion while TRA-1 induced onlya moderate apoptotic response and TRA-10 only induced a weak response.TRA-8 is therefore classified as an agonist anti-DR5 antibody. In FIG.4, the viability of human Jurkat cells is shown as a dose dependentfunction of TRAIL-induced apoptosis. TRA-10 blocked apoptosis of humanJurkat cells to a significant extent in a low dose TRAIL-inducedapoptosis study. Thus, TRA-10 is classified as an antagonist anti-DR5antibody. TRA-1 is on deposit with American Type Culture Collectionunder Accession Number PTA-1741. TRA-10 likewise was deposited on Apr.20, 2000, with American Type Culture Collection, 10801 UniversityBoulevard, Manassas, Va. 20110-2209 under Accession Number PTA-1742.

The susceptibility of five of the glioma cell lines. D-37MG, D54-MG,U373-MG, CH235-MG and 1321N1 to TRA-8-induced apoptosis is equivalent totheir susceptibility to TRAIL-mediated apoptosis as shown in FIG. 3b ,indicating that TRAIL-induced apoptosis in these cells is mediatedprimarily through DR5. Moreover, two of the glioma cell lines, Hs683 andU251-MG, are resistant to TRAIL-induced apoptosis but partiallysensitive to TRA-8-induced apoptosis, indicating that the decoyreceptors function in these cells and that use of the TRA-8 antibodybypassed this regulatory mechanism. In the prostate cancer cell lines,despite the varying sensitivity to apoptosis induced by TRA-8, thisparalleled the sensitivity of the cells to apoptosis induced by TRAIL,again suggesting that DR5 plays a major role of TRAIL-mediated apoptosisin the prostate cancer cells. Among the hematopoietic cells, it is foundthat Jurkat and CEM-6 are very susceptible to both TRA-8 andTRAIL-mediated apoptosis. The level of apoptosis induced by TRA-8 iscomparable to that induced by TRAIL as shown in FIGS. 2a and 3a ′. Onlyone of the glioma cell lines, U87, and two hematopoietic cell lines,U937 and Molt-4, exhibited sensitivity to TRAIL-induced apoptosis butare less sensitive or resistant to TRA-8-induced apoptosis. One cellline, the H-9 cell line, expressed detectable levels of DR5 but areresistant to apoptosis induced by either TRA-8 or TRAIL. While minimallevels of expression of DR5 are required for TRA-8-induced apoptosis,the level of expression of DR5 does not necessarily predict thesusceptibility of the cells to TRA-8 mediated apoptosis; decoy receptorsplay a role in modulating TRAIL-mediated apoptosis in some cells, butdoes not appear to play a major role in most of the cells tested todate; as anticipated the TRA-8 antibody bypasses the effects of thedecoy receptors; functional mutations of the DR5 receptor may occur intransformed cells; and, finally, intracellular regulatory mechanisms maybe as important, or more important than the decoy receptors in definingthe susceptibility of the cells to TRAIL and DR5-mediated apoptosis.

Previous studies have shown that the mRNA for DR5 is distributed widelyin normal tissues⁷. To evaluate the expression of DR5 at the proteinlevel, a panel of normal human tissue homogenates (Geno Technology. St.Louis, Mo.) is probed with the TRA-8 antibody in western blot analysis.Among nine normal human tissues, brain tissue is weakly positive (FIG.5a , lane 2). DR5 protein is not detectable by TRA-8 reactivity in liver(lane 1), lung (lane 3), kidney (lane 4), spleen (lane 5), testes (lane6), ovary (lane 7), heart (lane 8), or pancreas (lane 9). In contrast,all thirteen human cancer tissues stained positively with TRA-8 (FIG. 5b), including cancers of the ovary (lane 1), lung (lane 2), liver (lane3), rectum (lane 4), cervix (lane 5), skin (lane 6), testes (lane 7),thyroid (lane 8), uterus (lane 10), stomach (lane 11), laryngopharynx(lane 12), and pancreas (lane 13). Moreover, in situimmunohistochemistry of normal and cancer tissues with TRA-8 confirmedthat aside from a few scattered positive cells in spleen, DR5 expressionin normal breast, lung and spleen tissues is not detectable (FIG. 5c ).The corresponding cancer tissues including breast infiltrating ductalcarcinoma, small cell lung cancer, and lymphoma reacted positively withTRA-8 (FIG. 5d ). Among a total of 22 cancer tissues examined, 5 of 6breast cancers, 2 of 2 cancers of the cervix, 4 of 5 liver cancers, 5 of8 lymphomas, 2 of 2 lung cancers, and 2 of 2 prostate cancers reactedpositively with TRA-8. These results are consistent with those of theflow cytometry analysis and indicate that cancerous tissues expresshigher levels of DR5 protein than do normal tissues.

Example 9. Tumoricidal Activity of TRA-8 In Vivo

For various reasons, many agents that show promise in in vitro studiesdo not show efficacy in vivo). It is therefore important to test theefficacy of TRA-8 in an in vivo animal model. To accomplish this theTRA-8 anti-human DR5 antibody is administered to mice bearing humanxenografts that express the human DR5 molecule. The mice used are 6 to 8week-old NOD/SCID mice (Jackson Laboratory), which are inoculatedsubcutaneously with human astrocytoma 1321N1 cells (1×10⁷), orinoculated intravenously with human leukemia Jurkat cells (1×10⁶). Atday 2 after tumor inoculation, mice are inoculated intravenously withTRA-8 (100 μg). Five days after the treatment with TRA-8, 1321N1 tumorgrowth is determined by the size and weight of the tumor mass. Thegrowth of Jurkat cells is determined by the weight of the spleen and thepercentage of human CD3-positive Jurkat cells in the spleen ofinoculated animals. Biopsies of tumor tissues are taken and examinedhistologically.

Early treatment with a single intravenous dose of 100 μg of TRA-8 at oneday after tumor inoculation completely inhibited the 1321N1 cells fromforming a solid tumor of (FIG. 6a ). Late treatment with three doses of100 μg TRA-8 at one week after tumor inoculation reduced tumor weight4-fold or more (FIG. 6b ). Tumor formation is not visible in animalstreated with TRA-8 at an early time point (FIG. 6c , upper panel).Histologic analysis revealed dramatically degenerated tumor tissue inanimals treated with TRA-8 (FIG. 6c , lower panel). Similarly, TRA-8treatment inhibited population of the spleen by Jurkat cells asdemonstrated by the scarcity of CD3-positive Jurkat cells in the spleen(FIG. 6d, 6e ). Histological analysis of the implanted tumor showed afew tumor cells scattered in the soft tissue in TRA-8-treated animalswhile controls showed the formation of a solid tumor as shown in FIG. 6c. In the Jurkat cell model, the number of Jurkat cells in the spleens ofTRA-8 treated animals is less than 2% compared to nearly 10% in thespleen of control animals as demonstrated by flow cytometry analysis asshown in FIG. 6a and in situ CD3 staining of FIG. 6 c.

These results confirm the recent demonstration that systemicadministration of cross-linked recombinant TRAIL inhibits growth oftumor in viva (13). These results indicate that a single dose of TRA-8is highly effective in the elimination of tumor cells in vivo.

As an anti-human antibody is used in a murine model, the toxicity of theTRA-8 treatment could not be assessed. However, the study ofadministration of TRAIL in vivo indicated that no significant toxicityis associated with this treatment (13).

Example 10. RA Synovial Cells are Susceptible to TRAIL and TRA-8-InducedApoptosis

Most of the prior art studies of TRAIL-mediated apoptosis have focusedon malignant cells. TRAIL-mediated apoptosis according to the presentinvention is also therapeutic in autoimmune and inflammatory conditions,such as RA.

10.1 Flow Cytometric Analysis of the Expression of Cell Surface DR5 inRA Synovial Cells

The expression of DR5 on a panel of eight primary cultured synovialcells from patients with RA is compared with that on eight primarycultured synovial cells from patients with osteoarthritis (hereinafterreferred to as “OA”). The eight human primary RA synovial cell culturesRA-1014, RA-1016. RA-1021, RA-512, RA-707, RA-811, RA-716, and RA-929are kindly provided by Dr. M. Ohtsuki (Sankyo Co. Ltd., Tokyo, Japan)and cultured in DMEM supplemented with 10% FCS, penicillin,streptomycin, and glutamine. The seven OA synovial cell primary cellcultures are isolated from the synovial tissues of OA patients by astandard collagenase method and cultured under the same conditions. Thepassage number of all primary cells is under 10. The expression of DR5is determined by FACs analysis as described in Example 5.

All of the primary cultures of RA cells expressed high levels of surfaceDR5, and there is little variation in the expression levels among thesesynovial cells isolated from different patients as shown in FIG. 7a . Incontrast, the expression of surface DR5 on the surface of synovial cellsisolated from the OA patients is very low or undetectable as per FIG. 7b. SV40-transformed synovial cell are found to express high levels of DR5comparable with those exhibited by the RA cells. In contrast,non-transformed fibroblast cells expressed low levels of DR5 comparableto those exhibited by the OA cells in FIG. 7 b.

10.2 Susceptibility of RA Synovial Cells to Apoptosis Mediated by TRA-8or TRAIL

In general, all synovial cells isolated from the RA patients aresusceptible to both TRAIL and anti-DR5 antibody induced apoptosis, andall OA cells are resistant to TRAIL and anti-DR5 antibody inducedapoptosis as per FIG. 8a, b . These studies indicate that the TRA-8antibody targets altered cells in preference to normal cells. Moreover,the pattern of the susceptibility or resistance to apoptosis induced byTRAIL is correlated with that induced by anti-DR5 antibody, indicatingthat the synovial cells primarily utilize DR5 to trigger TRAILapoptosis.

As described for the malignant cells, the susceptibility to apoptosisinduced by TRAIL or anti-DR5 antibody varied among the RA synovial cellsalthough expressing similar levels of DR5. RA-512 and RA-707 are themost susceptible as over 80% cells are killed by concentrations of TRAILor TRA-8 below 20 ng/ml. RA-1014, RA-811, RA-716, and RA929 are amongthose with the intermediate susceptibility to TRAIL or TRA-8, withnearly 100% cell death occurring in the presence of high concentrations(>50 ng/ml) of TRAIL or TRA-8. In RA-1016 and RA1021 cells, although themajority (over 60%) of cells are killed by a low dose of TRAIL, orTRA-8, a portion of cells survived in the presence of highconcentrations of TRAIL or TRA-8, indicating that a sub-population ofcells are resistant to TRAIL-mediated apoptosis. In contrast, all OAcells are much less susceptible to TRAIL and TRA-8 induced apoptosis. Nogreater than 60% cells are killed in the OA52F and OA69F even in thepresence of high concentration of TRAIL or TRA-8. OA72M cells arecompletely resistant to TRAIL or TRA-8 induced apoptosis. The SV40transformed synovial cells are also susceptible to TRAIL and TRA-8induced apoptosis (data not shown). In contrast, the non-transformedfibroblast cells appeared to be resistant to TRAIL and TRA-8.

It has been shown previously that DR5 utilizes a FADD/caspase 8dependent pathway to trigger apoptosis (44). To determine thecaspase-dependence of DR5-mediated apoptosis of RA synovial cells, RAcells are cultured with TRAIL or anti-DR5 antibody in the presence ofspecific caspase inhibitors. Among eight caspase inhibitors tested,caspase 6, 8 and 10 inhibitors are able to inhibit apoptosis of RAsynovial cells induced by both TRAIL and DR5 as shown in FIG. 9,indicating that these three caspases are involved in DR5-mediatedapoptosis.

10.3 TRA-8 or TRAIL Induce NF-κb Activation in RA Synovial Cells withoutIncreased Release of MMPs

There is considerable evidence to support the concept that there areclose links between the signaling of apoptosis and the signaling ofproliferation (45). It has been established that DR5 is able to activatea NF-kb pathway in addition to apoptosis signaling transduction, andthat NF-κb activation may be able to transduce an anti-apoptosis signal.Therefore a gel-shift assay is carried out. Cells are stimulated with 50ng/ml of the recombinant soluble TRAIL, Fas ligand in the presence ofthe 1 mg/ml enhancer, or 50 ng/ml of TRA-8 for the indicated time. Thenuclear extracts are prepared and incubated with the double-stained[³²P]-labeled oligo-DNA probe. The results are analyzed using thecyclone phospha-imager (TopCount NXT, Packard Instrument Company, CT).After RA synovial cells are incubated with TNF-α or TRAIL, NF-κb isactivated in a time-dependent fashion. The TRA-8 antibody is able tostrongly activate NF-κb. In contrast, Fas ligand is unable to induceNF-κb activation.

Thus, although TRAIL and TRA-8 antibody induce a strong apoptosisresponse in RA synovial cells, they also activate NF-κb, and NF-κbactivation has been believed to contribute to the proinflammatory roleof TNF-α in RA. Thus, it is possible that TRAIL, like TNF-α, may serveas a pro-inflammatory cytokine. To determine whether there is a similarbiological consequence of NF-kb activation induced by TRAIL and TNF-α,the production of MMPs is determined by ELISA. Synovial cells arecultured in medium alone or with 50 ng/ml interleukin 1b, 10 ng/mlTNF-a, 50 ng/ml TRAIL, or 50 ng/ml TRA-8 overnight. The levels of theMMP-1 and MMP-3 in the culture supernatants are determined by the ELISAkits.

When RA synovial cells are incubated with a proinflammatory cytokine,TNF-α or IL-1b, the production of MMP-1, 3, and 13 is increased comparedto the medium control as shown in FIG. 10 b,c. In contrast, treatmentwith TRAIL or anti-DR5 antibody is not associated with increased releaseof these MMPs.

Example 11. A Failure to Induce Hepatocellular Toxicity

For 24-hour cell viability assays, fresh normal human hepatocytes in96-well plates were purchased from In Vitro Technology (Baltimore, Md.).The hepatocytes are cultured in the Hepatocyte Culture Medium containing1 μg/ml of either soluble TRAIL or TRA-8. For 6-hour viability assays,normal hepatocytes or hepatocellular cancer cells are isolated fromfresh surgical specimens collected from UAB Tissue Procurement Center.All reagents for isolation of human hepatocytes including hepatocytesperfusion buffer, digest medium, washing medium, and attachment mediumwere purchased from Gibco. The tissue slides are digested in theHepatocyte Digest Medium at 37° C. with shaking (50 rpm) for one hour.The isolated hepatocytes are harvested by low speed centrifugation (50g, 3 min), and washed with the Hepatocyte Washing Medium six times.Single cell suspension of hepatocytes are cultured in the AttachmentMedium containing 10% FCS in 96-well Matrigel plates (BD) for six hours.Non-attached hepatocytes are removed by twice washing with pre-warmedattachment medium. Attached hepatocytes are further incubated withvarious concentrations of soluble TRAIL or FasL, in the presence ofcrosslinker, or TRA-8 or CH11 for 6 hours.

TRAIL has at least two receptors (DR4 and DR5) that are capable ofinducing apoptosis. TRA-8 is used to determine whether crosslinking ofDR5 alone is sufficient to induce apoptosis of normal hepatocytes. DR5expression at the protein level is examined initially in five normalhuman liver tissues and five liver cancer tissues by in situimmunohistochemistry using TRA-8. Sections from the normal liver tissuesshowed normal architecture and cell morphology on H&E staining (FIG. 11a, left upper panels) in the absence of positive reactivity with TRA-8for DR5 (FIG. 11a , left lower panels). In contrast, the humanhepatocellular carcinoma tissue reacted positively with TRA-8 in apattern consistent with both cell membrane and cytoplasm presence of DR5on the cancerous cells. The human hepatocellular carcinoma cell lineHepG2 is also positive for DR5. These results are consistent among thefive normal liver tissues, and only one (liver adenoma) out of fiveliver cancer tissues is DR5-negative. These results are consistent withthe Western blot data, shown in FIG. 5a , that, as with other normaltissues, normal human liver tissue does not express significant levelsof DR5 protein. Furthermore. Western blot analysis of isolated, normalhuman hepatocytes probed with TRA-8 does not reveal detectable levels ofDR5.

Cell surface expression of DR5 on human hepatocytes by flow cytometryanalysis demonstrated that freshly prepared normal hepatocytes did notexpress detectable levels of cell surface DR5 (FIG. 11b , top leftpanels). Neither is it detected on normal human hepatocytes that hadbeen cryopreserved or placed in short-term culture. In contrast, freshlyisolated hepatocellular carcinoma cells as well as HepG2 cells expresscell surface DR5. Using Fas as a comparison, the normal hepatocytes,hepatocellular carcinoma cells, and HepG2 cells all expressed equivalentlevels of Fas (FIG. 11b , lower panels). These results are consistentwith those obtained using in situ immunohistochemistry and Western blotand indicate that cell surface DR5 is highly expressed in cancerousliver cells but not normal hepatocytes. The presence of mRNA levels forDR4, DR5, DcR1 and DcR2 in human hepatocytes, demonstrated by RT-PCR²³,suggests that human hepatocytes might express very low levels of DR5protein that are below the threshold for detection by TRA-8.

To determine whether TRA-8 induces hepatocellular toxicity, thesusceptibility of normal human hepatocytes to apoptosis induced by TRA-8and by soluble TRAIL plus crosslinker is examined. When normalhepatocytes are cultured in the presence of a high concentration ofTRAIL, a time-dependent decrease in cell viability is observed byATPLite® (FIG. 12a ) and MTT assays. TRAIL-mediated cell death of normalhepatocytes could be seen as early as four hours after addition ofTRAIL. At end of a 24-hour culture, more than 80% of the hepatocytes arekilled by TRAIL. In contrast, during the same culture period, TRA-8 didnot induce significant cell death in normal hepatocytes. The condensednuclei stained with Hoechst, a characteristic of apoptosis, areincreased in TRAIL-treated but not TRA-8-treated hepatocytes (FIG. 12b). The number of apoptotic hepatocytes is well correlated with decreasedcell viability as determined by ATPLite® assay, suggesting thatTRAIL-induced cell death of hepatocytes is mediated by apoptosis. Thisis confirmed by the ability of Z-VAD to inhibit TRAIL-mediated toxicityof hepatocytes. As cycloheximide is a potent apoptosis enhancer, theeffect of this compound on TRAIL and TRA-8-treated hepatocytes isinvestigated. During a four-hour culture, cycloheximide significantlyenhanced the cell death of hepatocytes induced by TRAIL, with greaterthan 70% hepatocytes being killed by TRAIL in the presence ofcycloheximide (FIG. 12c ). However, cycloheximide treatment is unable toenhance TRA-8-mediated cell death in hepatocytes. To compare thecharacteristics of apoptosis induced by TRA-8 with that induced by TRAILin hepatocytes, normal hepatocytes as well as cancer cells are incubatedwith variable concentrations of soluble TRAIL with crosslinker or TRA-8.During a 6-hour culture period. TRAIL induced a moderate apoptoticresponse in normal hepatocytes. Over 20% of hepatocytes are killed inthe presence of 500 ng/ml TRAIL (FIG. 12d , upper left). TRA-8-treatmentof normal hepatocytes did not elicit any significant cell death over thesame time period. In contrast to normal hepatocytes, primaryhepatocellular carcinoma cells (FIG. 12d , upper middle) and HepG2 cells(FIG. 12d , upper right) are highly susceptible to apoptosis mediated byeither TRAIL or TRA-8. Over 80% of hepatocellular carcinoma cells andnearly 100% of HepG2 cells are killed during the 8-hour culture period.These results indicate that normal hepatocytes are completely resistantto TRA-8-mediated apoptosis, and are much less susceptible toTRAIL-mediated apoptosis than are liver cancer cells. Using Fas ligandand anti-Fas antibody (CH-11), there is no significant difference in thesusceptibility to Fas-mediated apoptosis among normal hepatocytes,hepatocellular carcinoma cells, and HepG2 cells (FIG. 12d , lowerpanels).

Comparative Example 11B. Human Membrane-Bound TRAIL Induction ofHepatitis In Vivo

8-10 week-old female B6 mice are intravenously injected with 10⁹ pfu ofAd/h TRAIL with the equal number of Ad/Tet-on. Mice are fed withdifferent concentrations of tetracycline in their drinking waterimmediately after inoculation of adenoviral vectors. Liver injury isdetermined by serum levels of AST using an AST diagnostic kit (Sigma).Expression of TRAIL is determined by Northern blot analysis.

To determine whether the membrane bound form of TRAIL induces liverdamage in vivo, a recombinant adenoviral vector encoding the full lengthhuman TRAIL (Ad/hTRAIL) is constructed, the expression of which is underthe control of the tetracycline-inducible promoter. Twenty-four hoursafter intravenous inoculation of B6 mice with Ad/hTRAIL,tetracycline-induced expression of human TRAIL is observed in the liverin a dose-dependent fashion as demonstrated by Northern blot analysis(FIG. 13a ). The expression levels of TRAIL correlated well with liverdamage as shown by a tetracycline-dependent increase in serum levels oftransaminases, again in a dose-dependent fashion (FIG. 13b ). As theinoculation with adenoviral vector per se might increase thesusceptibility of hepatocytes to TRAIL-mediated apoptosis, thehepatocytes from mice inoculated with Ad/TRAIL are isolated and testedfor TRAIL-mediated cell death. There is no significantly increased celldeath of Ad/TRAIL infected hepatocytes compared to those from controlmice (FIG. 13c , left panel). Moreover. Ad/TRAIL inoculated mice did notexhibit increased liver injury after intravenous injection of solublehuman TRAIL. Thus, it follows that hepatitis induced by Ad/TRAIL ismediated by high levels of TRAIL expression in its membrane form.Histologic analysis of liver sections revealed that damage to thehepatocytes is apparent as early as 24 hours after vector inoculation(FIG. 13d ), and persisted for at least 7 days (FIG. 13e ). Thesepathologic alterations in the liver also are tetracycline-dependent andoccurred in a dose-dependent manner. The early phase, within 24 hours oftreatment, of TRAIL-induced liver damage is characterized by foci ofnecrosis. Infiltration of inflammatory cells is not observed at thisstage, but hemorrhage had occurred. By day 7 after inoculation, diffuseliver damage is apparent with marked lobular disarray, severedegeneration of hepatocytes with irregularly clumped cytoplasm and largeclear spaces, and prominent apoptosis and necrosis. An extensiveinfiltrate of mononuclear cells is a characteristic feature at thisstage. These results indicate that human TRAIL in its membrane-boundform is able to induce liver damage in vivo. Despite the propensity ofhuman TRAIL to cause severe hepatitis in mice, it did not induce alethal response. In contrast, mice inoculated with similartetracycline-controlled vectors encoding Fas ligand developed fulminanthepatitis with massive apoptosis and necrosis of hepatocytes accompaniedby severe hemorrhage and by mortality occurring in a tetracyclinedose-dependent within 72 hours of inoculation. The mortality ratereached 100% within 48 hours in those subgroups receiving 3 mg/ml ormore of tetracycline. In contrast, all of the mice that receivedAd/hTRAIL, regardless of the dose of tetracycline, are still alive fourweeks after inoculation. Thus, it follows that, in vivo, themembrane-bound form of TRAIL is a less potent inducer of hepatocellulardamage than Fas ligand. They further suggest that TRAIL might induceliver damage through a mechanism that differs from the mechanismunderlying the toxicity of Fas ligand.

Example 12. Activated Human T and B Cells Express Increased Levels ofDR5

To determine whether DR5 plays a role in TRAIL-mediated apoptosis ofactivated T cells and B cells, surface expression of DR5 on resting andactivated T and B cells using TRA-8 is examined. The unstimulated humanT cells in PBMC did not express significant levels of DR5 (FIG. 14). At48 hours after either anti-CD3 or Con-A stimulation, cell surface DR5expression is significantly increased. Similarly, the unstimulated Bcells expressed very low levels of DR5. Stimulation with anti-μ but notLPS resulted in increased cell surface expression of DR5. These resultsindicate that both activated T and B cells express higher levels of cellsurface DR5. Cells are stained with 20 μg/ml TRA-8 and PE anti-mouseIgG1.

Example 13. Activated T and B Cells Become Susceptible to TRA-8 MediatedApoptosis

To determine whether activated T and B cells are susceptible toTRA-8-mediated apoptosis, the T cells and B cells of human PBMC arestimulated with anti-CD3 or anti-μ in vitro for 48 hours, respectively.The viable cells and proliferating blast cells are collected by gradientcentrifugation, and incubated with various concentrations of TRA-8.Unstimulated T cells and B cells are not susceptible to TRA-8-mediatedapoptosis (FIG. 15). Total stimulated T cells and B cells showed amoderately increased susceptibility to TRA-8-mediated apoptosis, with20% cells being killed by TRA-8 after overnight culture. The highlyproliferating blast T cells are even more susceptible to TRA-8 mediatedapoptosis. More than 70% of the blast T cells could be killed by TRA-8.The blast B cells are also more susceptible to TRA-8 mediated apoptosiscompared to others. These results indicate that activated T and B cellsare susceptible to DR5-mediated apoptosis.

Example 14. TRA-8 Depletes Activated T Cells in Human/SCID Mice

To determine the in vivo anti-T cell efficacy of TRA-8, NOD/SCID miceare intravenously injected with 1×10⁸ human PBMC. Normally, the human Tcells in SCID mice are quickly activated in response to xenogeneicstimulation. The human PBMC/SCID mice are intraperitoneally injectedwith 100 μg TRA-8 or control IgG1 from the day of transfer, repeateddaily for three days. Five days after transfer, the mononuclear cellsare isolated from the spleen and stained with anti-human CD3 antibody,and the lymphocyte population is gated by flow cytometry analysis, andCD3 positive human T cells are analyzed. Approximately 30% of spleniclymphocytes are human T cells as determined by anti-human CD3 stainingin control treated mice. However, only a few human T cells (less than3%) are observed among the splenic lymphocytes in TRA-8 treated mice(FIG. 16). In situ histological study revealed that in the spleen ofcontrol mice, the human T cells are repopulated in the spleen with onlya few apoptotic cells observed as demonstrated by TUNEL staining. Incontrast, repopulation with viable human T cells is not observed in thespleen of TRA-8 treated mice, rather many apoptotic cells are observed(FIG. 17). These results demonstrate that TRA-8 has anti-T cell activityin vivo, and indicate the utility of the inventive antibodies for thetreatment of GVH disease.

Example 15. Anti-Cancer Therapeutic Activity of TRA-8 15.1 DR5Expression and Function in Human Cancer Tissues and Cell Lines

i) DR5 Expression in Human Cancer Tissues by In Situ Staining withTRA-8.

To determine whether cancer cells and tissues differentially expresshigher levels of DR5, a panel of human cancer tissues including over 20breast cancers, 6 ovarian cancers, 5 colon cancers and 5 prostatecancers are stained with TRA-8 for immunohistochemistry. The majority ofthese cancer tissues expressed detectable DR5. The expression levels ofDR5 in these cancer tissues varied. In general, cancer tissues expressedhigher levels of DR5 than uninvolved tissues. In addition, DR5expression is apparently not correlated with the mutation of p53.

ii) DR5 Expression and Function in Human Cancer Cell Lines (Table 2).

Nine human breast cancer cell lines, three ovarian cancer lines, threecolon cancer lines and three prostate cancer lines are examined for cellsurface expression of DR5 and susceptibility to TRA-8-induced apoptosisin vitro. 7 of 9 breast cancer lines, 3 of 3 ovarian cancer lines, 3 of3 colon cancer lines and 3 of 3 prostate cancer lines expressed variablelevels of cell surface DR5. Of 9 breast cancer lines, three are verysusceptible, three are intermediate and three are resistant toTRA-8-mediated apoptosis. All three ovarian cancer lines are verysusceptible. One of three colon cancer lines is very susceptible, whiletwo have intermediate sensitivity. Two of three prostate cancer lineshave intermediate sensitivity and one is resistant.

TABLE 2 Expression and function of DR5 in human cancer cells. Cell lineOrigin Expression¹ Susceptibility² 2LMP breast + ++++ LCC6 breast +++++++ MB468 breast +++ +++ MB231 breast ++ +++ ZR-75-1 breast +++ ++SKBR3 breast + ++ MB453 breast ++ + BT474 breast + − DY36T2 breast − −Caov-3 ovary + ++++ OVCAR-3 ovary ++ ++++ Skov-3 ovary + +++ WiDR colon+++ ++++ HST29 colon ++ +++ T84 colon + ++ PC3 prostate +++ ++ LnCapprostate +++ + Du-145 prostate +++ + Note: ¹determined by flowcytometry, cells are stained with 20 μg/ml TRA-8 and compared to controlantibody. ²determined by ATPLite ® assay. ++++: over 80% killing, +++:killing between 60-80%, ++: killing between 40-60%, +: killing between20-40%, − no killing.

iii) Combined Cytotoxicity of TRA-8 with Adriamycin.

In several breast cancer lines, the effect of adriamycin on TRA8-inducedapoptosis is examined. High doses of adriamycin exhibited an additiveeffect. However, in some of TRA-8 resistant lines, low doses ofadriamycin synergistically enhance TRA-8-induced apoptosis.

iv) In Vitro and In Vivo Binding Activity of TRA-8 to Human CancerCells.

Using radioisotope labeled TRA-8. The binding activity of TRA-8 to abreast cancer line is examined in vitro and in viva in SCID miceimplanted with tumor. The in vitro binding activity to cancer cells isestimated as a Kd value of 3 nM, which is constant with our previousestimation using ELISA, and at least 50-fold higher than soluble TRAIL.In vivo, TRA-8 localized to implanted tumor tissues.

15.2. Therapy of Chronic Lymphocytic Leukemia in NOD/SCID Mice withTRA-8

Chronic lymphocytic leukemia (CLL) is a common form of B cellmalignancy. Most malignant B cells in CLL are of the mature phenotypeand are resistant to many apoptosis stimuli. DR5 expression and functionin the B cells of five patients with CLL is examined. All patients hadhigh counts of peripheral B cells as shown by more than 95% CD19+ Bcells in PBMC. Compared to normal primary B cells, the CLL B cells ofall patients had higher levels of cell surface DR5 and are moresusceptible to TRA-8 induced apoptosis in vitro. Interestingly, the CLLB cells are also sensitive to bisindolemaleimide VIII (BisVIII) inducedcytotoxicity. Following combined treatment with TRA-8 and BisVIII,nearly 50% of CLL B cells are killed white normal B cells remainedunresponsive (FIG. 18). Transfer of CLL B cells into NOD/SCID miceresulted in about 25/%-30% CD19+ B cells repopulated in the spleen ofrecipient mice at five days after transfer. However, three doses of 100μg TRA-8 treatment completely eliminated CLL B cells of four out of fivepatients in the spleen of the recipient SCID mice. Thus, TRA-8 alone orin concert with other substances is active as a therapeutic agent forchronic lympholytic leukemia.

Example 16. cDNA Cloning

(1) Determination of the N-Terminal Amino Acid Sequences of the Heavyand Light Chains of TRA-8

In order to obtain cDNAs of the heavy and light chains of TRA-8, theN-terminal amino acid sequences of the heavy and light chains of TRA-8and cloned TRA-8 genes are determined by known techniques.

Ten μg of the solution containing the anti-human DR5 antibody TRA-8 issubjected to SDS-polyacrylamide gel electrophoresis (“SDS-PAGE”), usinga gel concentration of 12% w/v, 100 V constant voltage, for 120 minutes.After electrophoresis, the gel is immersed in transfer buffer 25 mMTris-HCl (pH 9.5), 20% methanol, 0.02% v/v SDS for 5 minutes. After thistime, the protein content of the gel is transferred to a polyvinylidenedifluoride membrane (“PVDF membrane”; pore size 0.45 um: Millipore,Japan), presoaked in transfer buffer, using a blotting apparatus(KS-8451; Marysol) under conditions of 10 V constant voltage, 4° C., for14 hours.

After this time, the PVDF membrane is washed with washing buffer 25 mMNaCl, 10 mM sodium borate buffer (pH 8.0), then stained in a stainingsolution (50% v/v methanol, 20% v/v acetic acid and 0.05% w/v CoomassicBrilliant Blue) for 5 minutes to locate the protein bands. The PVDFmembrane is then destained with 90% v/v aqueous methanol, and the bandscorresponding to the heavy chain, the band with the lower mobility andlight chain, the band with the higher mobility previously located on thePDVF membrane are excised and washed with deionized water.

The N-terminal amino acid sequence of the heavy and light chains aredetermined by the Edman automated method (Edman, P., et al., (1967),Eur. J. Biochem., 1, 80) using a gas-phase protein sequencer (PPSQ-10;Shimadzu Seisakusyo, K. K.).

The N-terminal amino acid sequence of the band corresponding to theheavy chain is determined to be:

(SEQ ID No. 4 of the Sequence Listing)Glu-Val-Met-Leu-Val-Glu-Ser-Gly-Gly-Gly-Leu-Val-Lys-Pro-Gly-Gly-Ser-Leu-Lys-Leu;and that of the band corresponding to the light chain is determined tobe:

(SEQ ID No. 5 of the Sequence Listing)Asp-Ile-Val-Met-Thr-Gln-Ser-His-Lys-Phe-Met-Ser-Thr-Ser-Val-Gly-Asp-Arg-Val-Ser.

Comparison of these amino acid sequences with the database of amino acidsequence of antibodies produced by Kabat et al. (Kabat E. A., et al.,(1991), in “Sequences of Proteins of Immunological Interest Vol. II,”U.S. Department of Health and Human Services) revealed that the heavychain (γ1 chain) and the light chain (k chain) of TRA-8 belonged tosubtypes 3d and 1, respectively.

(2) cDNA Cloning

Based on above findings, oligonucleotide primers are synthesized whichwould be expected to hybridize with portions of the 5′-untranslatedregions and the very ends of the 3′-translated regions of the genesbelonging to these mouse subtypes. Then, cDNAs encoding the heavy andlight chains of TRA-8 are cloned by the following combination of reversetranscription and PCR (RT-PCR):

a) Template

The total RNA of TRA-8 hybridoma (ATCC No. PTA-1428) is extracted byusing TRIzol Reagent (GIBCO BRL). The template for the PCR reaction usedcDNA that is obtained by using the First-Strand cDNA synthesis kit(Amersham Pharmacia Biotech) according to the instruction manualprovided with the kit.

b) PCR Primers

The following oligonucleotide primers are synthesized for the PCR:

(H5NCS1: SEQ ID No. 6 of the Sequence Listing)5′-cagcactgaa cacggacccc-3′;(H5NCS2: SEQ ID No. 7 of the Sequence Listing)5′-aaaggtaatt tattgagaag-3′;(H5SS1: SEQ ID No. 8 of the Sequence Listing)5′-cctcaccatg aacttcgggc-3′;(H5SS2: SEQ ID No. 9 of the Sequence Listing)5′-ctgttgtatg cacatgagac-3′;(H5CS1: SEQ ID No. 10 of the Sequence Listing)5′-gaagtgatgc tggtggagtc-3′;(H5CS2: SEP ID No. 11 of the Sequence Listing)5′-agtgtgaagt gatgctggtg-3′;(H3CR: SEQ ID No. 12 of the Sequence Listing)5′-tttaccagga gagtgggaga g-3′;(H3VR: SEQ ID No. 13 of the Sequence Listing)5′-tgcagagaca gtgaccagag-3′; (L5NCS1: SEQ ID No. 14 of theSequence Listing) 5′-tgttcaggac cagcatgggc-3′;(L5NCS2: SEQ ID No. 15 of the  Sequence Listing)5′-aagacatttt ggattctaac-3′;(L5SS1: SEQ ID No. 16 of the Sequence Listing)5′-tatcatgaag tctttgtatg-3′;(L5SS2: SEQ ID No. 17 of the Sequence Listing)5′-gatggagaca cattctcagg-3′;(L5CS: SEQ ID No. 18 of the Sequence Listing)5′-gacattgtga tgacccagtc-3′;(L3CR: SEQ ID No. 19 of the Sequence Listing)5′-ttaacactca ttcctgtga-3′; and(LCSR: SEQ ID No. 20 of the Sequence Listing)5′-gactgggtca tcacaatgtc-3′.

Unless otherwise specified, all oligonucleotides in these Examples aresynthesized by Pharmacia Biotech. All oligonucleotides are stored at−20° C. after being dissolved in distilled water.

c) PCR Reaction

Composition of the PCR reaction solution:

template cDNA, 5 μl of total 33 μl reaction

primer DR5p1, 10 pmol;

primer DR5p2, 10 pmol;

10× concentrated PCR buffer (provided with the kit), 10 μl;

dNTPs (each 2.5 mM), 4 μl; and

Taq polymerase (Promega), 5 units.

Sterile distilled water is added to the solution to a total volume of100 μl. Unless otherwise specified, dNTPs are an equimolar mixture ofdATP, dCTP, dGTP and dTTP (2.5 mM each).

The PCR reaction is conducted as follows. The solution is first heatedat 94° C. for 2 minutes, after which a cycle of heating to 94° C. for 30see, 52° C. for 1 minute and 72° C. for 3 minutes, is repeated 40 times.After completion of this procedure, the reaction solution is heated at72° C. for 10 minutes.

The amplified DNA fragments, thus obtained, are separated on a 1%agarose gel containing 0.25 μg/ml ethidium bromide. The bands determinedto contain the desired DNA fragments are cut out using a razor blade andthe DNA is recovered therefrom using the Gene Clean kit (BIO101). TheDNA fragment is cloned using pGEM-T Easy vector (Promega). This isperformed as follows.

The DNA fragment recovered from the PCR reaction solution, together with50 ng of pGEM-T Easy vector (provided with the kit), is mixed with 1 μlof 10× ligase reaction buffer (6 mM Tris-HCl (pH 7.5), 6 mM magnesiumchloride, 5 mM sodium chloride, 7 mM β-mercaptoethanol, 0.1 mM ATP, 2 mMDTT, 1 mM spermidine, and 0.1 mg/ml bovine serum albumin), to which 4units of T4 DNA ligase (1 μl) has been added. The total volume of themixture is adjusted to 10 μl with sterile deionized water, and theresulting ligase solution is incubated at 14° C. for 15 hours. Afterthis time, 2 μl of the ligase reaction solution is added to 50 μl ofcompetent E. coli strain JM109 (provided with the kit and brought tocompetence in accordance with the instruction manual) to which 2 μl of0.5 M β-mercaptoethanol had been added, and the resulting mixture iskept on ice for 30 minutes, then at 42° C. for 30 seconds, and again onice for 5 minutes. Next, 500 μl of medium containing 2% v/v tryptone,0.5% w/v yeast extract, 0.05% w/v sodium chloride, 2.5 mM potassiumchloride, 1 mM magnesium chloride, and 20 mM glucose (hereinafterreferred to as “SOC” medium) is added to the culture, and the mixture isincubated for 1 hour at 37° C. with shaking. After this time, theculture is spread on an L-broth agar plate (1% v/v tryptone, 0.5% w/vyeast extract, 0.5% w/v sodium chloride, 0.1% w/v glucose, and 0.6% w/vbacto-agar (Difco)), containing 100 g/ml. Ampicillin resistant coloniesappearing on the plate are selected and scraped off with a platinumtransfer loop, and cultured in L-broth medium containing 100 μg/mlampicillin at 37° C., overnight, with shaking at 200 r.p.m. Afterincubation, the cells are harvested by centrifugation, from whichplasmid DNA is prepared by the alkali method. The obtained plasmid isdesignated as plasmid pH62 for heavy chain of TRA-K or pL28 for lightchain of TRA-8. The transformant E. coli strains harboring theseplasmid, designated as E. coli JM109/pH62 and E. coli JM109/pL28 weredeposited with International Patent Organism Depositary, NationalInstitute of Advanced Industrial Science and Technology, 1-1, Higashi 1chome Tsukuba-shi, Ibaraki-ken, 305-5466, Japan on Apr. 20, 2001, inaccordance with the Budapest Treaty for the Deposit of Microorganisms,and were accorded the accession numbers FERM BP-7560 and FERM BP-7561,respectively. The nucleotide sequences of these DNAs encoding the heavychain and the light chain of TRA-8 are confirmed by the dideoxy method(Sanger, F. S., et al., (1977), Proc. Natl. Acad. Sci. USA,74:5463-5467) using 3700 DNA Analyzer (ABI PRISM®; Perkin Elmer AppliedBiosystems, Japan).

The nucleotide sequences of the heavy and light chains of TRA-8 aregiven as SEQ ID No. 21 and No. 22 of the Sequence Listing, respectively.The amino acid sequences of the heavy and light chains of TRA-8 aregiven as SEQ ID No. 23 and No. 24 of the Sequence Listing, respectively.The N-terminal amino acid sequences of the heavy and light chains ofTRA-8 established in above matched perfectly. Furthermore, when theamino acid sequences of the heavy and light chains are compared with thedatabase of amino acid sequences of antibodies, it is established that,for the heavy chain, nucleotide Nos. 58 to 414 in SEQ ID No. 21constituted the variable region, while nucleotide Nos. 415 to 1392 inSEQ ID No. 21 constituted the constant region. For the light chain,nucleotide Nos. 64 to 387 in SEQ ID No. 22 constituted the variableregion, while nucleotide Nos. 388 to 702 in SEQ ID No. 22 constitutedthe constant region. The locations and sequences of the CDRs are alsoelucidated by comparing the homologies with the database. The amino acidsequences of CDR1, CDR2, and CDR3 of heavy chain of TRA-8 are shown inSEQ ID No. 25, No. 26, and No. 27, respectively. The amino acidsequences of CDR1, CDR2, and CDR3 of light chain of TRA-8 are shown inSEQ ID No. 28, No. 29, and No. 30, respectively.

Example 17. Designing a Humanized Version of the TRA-8 Antibody

(1) Molecular Modeling of a Variable Region of TRA-8

Molecular modeling of the variable region of TRA-8 is performed by themethod generally known as homology modeling (Methods in Enzymology, 203,121-153, (1991)). The primary sequences of variable regions of humanimmunoglobulin registered in the Protein Data Bank (Nuc. Acid Res. 28,235-242 (2000)), for which the three-dimensional structures derived fromx-ray crystallography are available, are compared with the frameworkregions of TRA-8 determined above. As a result, 1NCD and 1HIL areselected as having the highest sequence homologies to the frameworkregions for the light and heavy chains of TRA-8, respectively.Three-dimensional structures of the framework regions are generated bycombining the coordinates of 1NCD and 1HIL which correspond to the lightand heavy chains of TRA-8, to obtain the “framework model”. Using theclassification defined by Chothia et al., the CDRs of TRA-8 areclassified as follows; CDRL₁, CDRL₇, CDRH₁ and CDRH₂ belong to canonicalclasses 2,1,1,3, respectively, while CDRL₃ does not belong to anyspecific canonical classes. The CDR loops of CDRL₁, CDRL₂, CDRH₁, CDRH₂are fixed to the conformations inherent to their respective canonicalclasses, and integrated into the framework model. CDRL₃ is assigned theconformation of cluster 8A, according to the classification of Thorntonet al. (J. Mol. Biol., 263, 800-815, (19%)), and CDRH₃ is classifiedinto k(8)C using the H3 rule (FEBS letter 455, 188-197 (1999)). Thenrepresentative conformations for CDRL₃ and CDRH₃ are integrated into theframework model.

Finally, energy calculations are carried out to eliminate unfavorableinter-atomic contacts, in order to obtain a probable molecular model ofTRA-8's variable region in terms of energy. The above procedure isperformed using the commercially available common molecular modelingsystem ABM (Oxford Molecular Limited. Inc.). For the molecular modelobtained, the accuracy of the structure is further evaluated using thesoftware, PROCHECK (J. Appl. Cryst. (1993), 26, 283-291).

(2) Designing the Amino Acid Sequences for Humanized TRA-8.

Construction of humanized TRA-8 antibodies is performed by the methodgenerally known as CDR grafting (Proc. Natl. Acad. Sci. USA 86,10029-10033 (1989)). The acceptor antibody is chosen based on the aminoacid homology in the framework region. The sequences of framework regionin TRA-8 are compared with all the human framework sequences in theKabat database of amino acid sequences of antibodies (Nuc. Acid Res. 29,205-206 (2001)). As a result, mAB58′CL antibody is selected as anacceptor due to the highest sequence homology of 80% for the frameworkregion. The amino acid residues in the framework region for mAb58′CL arealigned with that for TRA-8 and the positions where different aminoacids are used are identified. The location of those residues areanalyzed using the three dimensional model of TRA-8 constructed aboveand the donor residues which should be grafted on the acceptor arechosen by the criteria given by Queen et al. (Proc. Natl. Acad. Sci. USA86, 10029-10033 (1989)). Humanized TRA-8 sequences are constructed asdescribed in the following example by transferring several donorresidues into acceptor antibody, mAb58′CL.

Example 18. Construction of an Expression Vector for the Heavy Chain ofthe Humanized Antibody

(1) Construction of Plasmid Carrying the Heavy Chain Variable Region DNAof Humanized TRA-8

In order to determine the activity of humanized TRA-8, the plasmidcarrying the heavy chain of humanized TRA-8 is constructed as follows.However, it is appreciated the humanization of TRA-8 is not limited tothese examples.

As shown in SEQ ID No. 31 of the Sequence Listing, humanization of theamino acid sequences of the heavy chain of the mouse anti-human DR5antibody TRA-8 entailed replacing the 13th amino acid (lysine), the 19thamino acid (lysine), the 40th amino acid (threonine), the 42nd aminoacid (glutamic acid), the 44th amino acid (arginine), the 84th aminoacid (serine), the 88th amino acid (serine), the 93rd amino acid(methionine), the 114th amino acid (threonine), the 115th amino acid(leucine) with glutamine, arginine, alanine, glycine, glycine,asparagine, alanine, valine, leucine, and valine, respectively.

The plasmid carrying DNA encoding heavy chain variable region ofhumanized TRA-8 (SEQ ID No. 31 of the Sequence Listing) are constructedas follows.

PCR is used to construct the following DNA sequences, each of whichcomprised described above:

The following 12 oligonucleotides are synthesized:

(A; SEQ ID No. 32) 5′- ttggataagc ttggcttgac ctcaccatgg gatggagctgtatcatcctc ttcttggtag caacagctac aggtgtccac -3′; (B; SEQ ID No. 33)5′- tctgaagtaa tgctggtgga gtctggggga ggcttagtacagcctggagg gtccctgaga ctctcctgtg cagcctctgg -3′; (C; SEQ ID No. 34)5′- attcactttc agtagttatg taatgtcttg ggttcggcaggcaccaggga agggtctgga gtgggttgca accattagta -3′; (D; SEQ ID No. 35)5′- gtggtggtag ttacacctac tatccagaca gtgtgaagggccgattcacc atctccagag acaatgccaa gaacaccctg -3′; (E; SEQ ID No. 36)5′- tatctgcaaa tgaacagtct gagagcagag gacacggctgtttattactg tgcaagaagg ggtgactcta tgattacgac -3′; (F; SEQ ID No. 37)5′- ggactactgg ggccaaggga ccctggtcac agtctcctca gcctc cacc aagggcccat cggtc -3′; (G; SEQ ID No. 38)5′- ctaccaagaa gaggatgata cagctccatc ccatggtgag gtcaagccaa gcttatccaa -3′; (H; SEQ ID No. 39)5′- tctcagggac cctccaggct gtactaagcc tcccccagac tccaccagca ttacttcaga gtggacacct gtagctgttg -3′; (I; SEQ ID No. 40)5′- tccagaccct tccctggtgc ctgccgaacc caagacattacataactact gaaagtgaat ccagaggctg cacaggagag -3′; (J; SEQ ID No. 41)5′- ctctggagat ggtgaatcgg cccttcacac tgtctggatagtaggtgtaa ctaccaccac tactaatggt tgcaacccac -3′; (K; SEQ ID No. 42)5′- ccttcttgca cagtaataaa cagccgtgtc ctctgctctcagactgttca tttgcagata cagggtgttc ttggcattgt -3′; and (L; SEQ ID No. 43)5′- gaccgatggg cccttggtgg aggctgagga gactgtgaccagggtccctt ggccccagta gtccgtcgta atcatagagt cacc -3′.The following 2 PCR primers are synthesized as described above:(P1; SEQ ID No. 44) 5′- ttggataagc ttggcttgac -3′; and(P2; SEQ ID No. 45) 5′- gaccgatggg cccttggtgg a -3 ′.

The synthesis of DNA encoding a polypeptide chain comprising a secretionsignal sequence, a variable region of humanized TRA-8 heavy chain andthe 8 amino acid residues at the N-terminus of the IgG-CH1 region isperformed using a combination of PCR respectively.

The DNA fragment is prepared as follows.

Composition of the PCR reaction solution:

oligonucleotide A, 10 pmol;

oligonucleotide B, 10 pmol;

oligonucleotide C, 10 pmol;

oligonucleotide D, 10 pmol;

oligonucleotide E, 10 pmol;

oligonucleotide F, 10 pmol;

oligonucleotide G, 10 pmol;

oligonucleotide H, 10 pmol;

oligonucleotide I, 10 pmol;

oligonucleotide J, 10 pmol;

oligonucleotide K, 10 pmol;

oligonucleotide L, 10 pmol;

oligonucleotide primer P1, 2 μM;

oligonucleotide primer P2, 2 μM;

10× Pyrobest™ buffer II, 10 μl;

dNTP mix, 8 μl;

Pyrobest™ DNA polymerase, 0.5 μl; and

Redistilled water to a final volume of 50 μl.

The PCR reaction is conducted as follows. The solution is first heatedat 94° C. for 5 minutes, after which a cycle of heating to 98° C. for 10second, 55° C. for 30 second and 72° C. for 1 minute, is repeated 7times. After completion of this procedure, the reaction solution isheated at 72° C. for 15 minutes.

An equal volume of phenol-chloroform (50% v/v phenol saturated withwater, 48% v/v chloroform, 2% v/v isoamyl alcohol) is added to 200 μl ofeach of the PCR products, and vigorously mixed for 1 minute. After thistime, the mixture is centrifuged at 10,000×g, and the aqueous layer isrecovered and mixed with an equal volume of chloroform-isoamyl alcohol(96% v/v chloroform and 4% v/v isoamyl alcohol), which is againvigorously at 10,000×g and the aqueous layer is recovered. The series ofsteps recited in this paragraph is referred to, hereafter, as “phenolextraction”).

Ethanol precipitation is then performed on the recovered aqueous layer.As used and referred to herein, “ethanol precipitation” consists ofadding, with mixing, a one tenth volume of 3M sodium acetate (pH 5.2)and 2.5 volumes of 100% ethanol to the solution to be treated, andfreezing the mixture using dry ice. The resulting mixture is thencentrifuged at 10,000×g to recover DNA as a precipitate.

After phenol extraction and ethanol precipitation, the resulting DNAprecipitate is vacuum-dried, dissolved in a minimum of redistilledwater, and separated by 3% agarose gel electrophoresis. Afterelectrophoresis, the gel is stained with a 1 μg/ml aqueous solution ofethidium bromide to allow detection of DNA under UV light. The DNA bandcorresponding to humanized TRA-8 DNA is cut out using a razor blade andeluted from the gel using Geneclean® Spin Kit (BIO 101, CA, USA). Afterphenol extraction, the eluted DNA is then concentrated by centrifugationat 7,500×g, followed by ethanol precipitation, and finally dissolved in5 μl of distilled water.

The resulting, each extracted DNA is cloned using pGEM-T Easy vector(Promega) as follows:

The DNA fragment recovered from the PCR reaction, 5 μl;

10× Taq polymerase buffer, 1 μl;

dNTP mixture, 1 μl

Taq polymerase (5 unit/ml), 1 μl; and

redistilled water to a final volume of 10 μl.

After the above each solution is reacted at 70° C. for 30 minutes, eachDNA solution and pGEM-T Easy vector are ligated using a DNA Ligation KitVersion 2.0 (Takara Shuzo Co., Ltd.) using the manufacturer's protocol.

After 4 hours incubation at 15° C., 2 μl of the incubated reactionsolution is mixed with 100 μl of competent E. coli strain JM109 at acell density of 1-2×10⁹ cells/ml (Takara Shuzo Co., Ltd.), and themixture is kept on ice for 30 minutes, then at 42° C. for 30 seconds,and again on ice for 1 minutes. Then, 500 μl of SOC medium (2% v/vtryptone, 0.5% w/v yeast extract, 0.05% w/v sodium chloride, 2.5 mM w/vpotassium chloride, 1 mM magnesium chloride, and 20 mM glucose) is addedthe mixture, which is incubated for a further hour, with shaking.Transformant strains are then isolated, and plasmid DNA is prepared fromthe strains as described in “Molecular Cloning A Laboratory Manual”. Thenucleotide sequences of these DNAs encoding the heavy chain of humanizedTRA-8 are confirmed by the dideoxy method (Sanger, F. S., et al.,(1977). Proc. Natl. Acad. Sci. USA, 74:5463-5467) using 3700 DNAAnalyzer (ABI PRISM®; Perkin Elmer Applied Biosystems, Japan).

The resulting plasmids are designated pHB14 (the plasmid carrying cDNAencoding the heavy chain of humanized TRA-8). The transformant E. colistrain harboring these plasmid, designated as E. coli JM109/pHB14 wasdeposited with International Patent Organism Depositary, Nationalinstitute of Advanced Industrial Science and Technology, 1-1, Higashi 1chome Tsukuba-shi, Ibaraki-ken, 305-5466, Japan on Apr. 20, 2001, inaccordance with the Budapest Treaty for the Deposit of Microorganisms,and was accorded the accession number FERM BP-7556.

(2) Construction of Expression Plasmids Carrying the Heavy ChainVariable Region DNA of Humanized TRA-8

Recombinant expression vectors for animal cells are constructed byinserting the DNA encoding the heavy chain of humanized TRA-8 (cloned inabove) as follows.

One μg of plasmid pSRHHH3 (European patent application EP 0-909-816-A1)carrying the heavy chain variable region of humanized anti-Fasmonoclonal antibody HFE7A and human IgG1 constant region genomic DNA, anexpression vector for mammalian cells, is digested with the restrictionenzymes HindIII and ApaI, and separated by 3% agarose gelelectrophoresis. After electrophoresis, the gel is stained with a 1μg/ml aqueous solution of ethidium bromide to allow detection of DNAunder UV light. The vector DNA bands containing human IgG1 constantregion genomic DNA without the heavy chain variable region of humanizedHFE7A are cut out using a razor blade and eluted from the gel usingGeneclean® Spin Kit (BIO 101, CA, USA). After phenol extraction, theeluted DNA is then concentrated by centrifugation at 7.500×g, followedby ethanol precipitation, and finally dissolved in 5 μl of distilledwater and then dephosphorylated using CIP. The resulting digested,dephosphorylated plasmid (100 ng) is ligated with 1 μg of the pHB14 DNAfragment containing the DNA encoding the heavy chain variable region ofhumanized TRA-8, which had also been digested with HindIII and ApaI,using a DNA Ligation Kit Version 2.0 (Takara Shuzo Co., Ltd.). Theligation mixture is then used to transform E. coli JM109, which is thenplated on LB agar plates containing 50 μg/ml ampicillin.

The transformants obtained by this method are cultured in 2 ml of liquidLB medium containing 50 μg/ml ampicillin at 37° C. overnight, andplasmid DNA is subsequently extracted from the resulting culture by thealkaline-SDS method.

The extracted plasmid DNA is digested with HindIII and ApaI, andsubjected to 3% w/v agarose gel electrophoresis to confirm the presenceor absence of the insert of the DNA encoding the heavy chain variableregion of humanized TRA-8. The insertion and orientation of the desiredDNA fragment in the vector is confirmed by DNA sequencing using a genesequence analyzer (ABI PRISMO® 3700 DNA Analyzer; Applied Biosystems).The resulting expression plasmid carrying cDNA encoding the heavy chainof humanized TRA-8 is designated pHB14-1.

Example 19. Construction of an Expression Vector for the Light Chain ofthe Humanized Antibody

(1) Construction of Vectors for the Light Chains of Humanized Versionsof TRA-8 Antibody

As shown in SEQ ID No. 46 of the Sequence Listing, in humanizing theamino acid sequence of the light chain of the mouse anti-human DR5antibody TRA-8, 8th amino acid (histidine), 9th amino acid (lysine),10th amino acid (phenylalanine), 11th amino acid (methionine), 13thamino acid (threonine), 20th amino acid (serine), 42nd amino acid(glutamine), 43rd (serine), 60th amino acid (aspartic acid), 63rd aminoacid (threonine), 77th amino acid (asparagine), 78th amino acid(valine), 80th amino acid (serine) 83rd amino acid (leucine), 85th aminoacid (aspartic acid), 87th amino acid (phenylalanine), and 99th aminoacid (glycine) 103rd amino acid (leucine) and 108th amino acid (alanine)from the N-terminus of the amino acid sequence of the TRA-8 light chainare replaced with proline, serine, serine, leucine, alanine, threonine,lysine, alanine, serine, serine, serine, leucine, proline,phenylalanine, threonine, tyrosine, glutamine, valine and threoninerespectively. The resulting sequence is designated LM2.

Expression plasmids carrying this type of humanized light chain aminoacid sequences of the anti-human DR5 antibody TRA-8 is constructed asfollows.

1) Synthesis of Primers for Preparing the Variable and Constant Regionsof the Light Chain of Humanized TRA-8

DNA coding for the LM2 polypeptide chain (SEQ ID No. 46 of the SequenceListing), each of which is a fusion of the variable region of humanizedanti-DR5 antibody TRA-8 light chain and the constant region of the humanIg light chain (κ chain), are respectively synthesized by usingcombinations of PCR.

Further to 7AL1P (SEQ ID No. 47) and 7ALCN (SEQ ID No. 48), thefollowing oligonucleotide primers are synthesized for PCR:

(HKSPR11; SEQ ID No. 49)5′- gtcccccaca gatgcagaca aagaacttgg agattgggtc atcacaatgt caccagtgga -3′; (HKCDF11; SEQ ID No. 50)5′- ccaagttctt tgtctgcatc agtaggagac agggtcacca tcacctgc -3′;(HKCDR12; SEQ ID No. 51) 5′- agtgtgccgg gtggatgccc agtaaatcag tagtttaggagatttccctg gtttctg -3′; (HKCDF22; SEQ ID No. 52)5′- tgggcatcca cccggcacac tggggtccca agcaggttta gtggcagt -3′;(HKCDR22; SEQ ID No. 53)5′- ataactacta tattgctgac agtaataggt tgcaaaatcc tccggctgca gactagagat ggt -3′; and (HKCF12; SEQ ID No. 54)5′- cagcaatata gcagctatcg gacgttcggt caaggcacca aggtggaaat caaacggact gtg -3′.

2) Construction of plasmid pCR3.1/M2-1 (Cloning of Humanized TRA-8 LightChain)

LM2-DNA fragment as defined in SEQ ID No. 55 of the Sequence Listingcoding for the amino acid sequence as defined in SEQ ID No. 46 of thesame is prepared by performing 2-step PCR, inserted into a plasmidvector and cloned in E. coli.

a) First Step PCR

LM2-F1-DNA fragment coding for a secretion signal sequence and a portionof FRL₁ region with a Hind III restriction enzyme cleavage site added atthe 5′-end is prepared under the following conditions. The templateplasmids, pHSGHM17 and pSRPDHH, are obtained by following thedescription in a European patent application EP 0 909 816 A1.

Composition of the Reaction Solution:

plasmid pHSGHM17 DNA (European patent application EP 0 909 816 A1), 25ng

oligonucleotide primer 7AL1P, 50 pmol

oligonucleotide primer HKSPR11, 50 pmol

dNTPs cocktail, 5 μl

10×PCR buffer, 5 μl

ampliTaq DNA polymerase (PerkinElmer), 2.5 units

The reaction solution having the above composition is adjusted to afinal volume of 50 μl by adding redistilled water and used in PCR.

PCR thermal conditions: Heating at 94° C. for 2 minutes, after which athermal cycle of 94° C. for 1 minute, 55° C. for 1 minute and 72° C. for2 minutes, repeated 30 times, followed by heating at 72° C. for 10minutes.

LM2-F2-DNA fragment coding for a portion of FRL₁, CDRL₁, FRL₂, and CDRL₂and is prepared under the following conditions.

Composition of the Reaction Solution:

plasmid pL28 DNA, 25 ng

oligonucleotide primer HKCDF11, 50 pmol

oligonucleotide primer HKCDR12, 50 pmol

dNTPs cocktail, 5 μl

10×PCR buffer, 5 μl

ampliTaq DNA polymerase, 2.5 units

The reaction solution having the above composition is adjusted to afinal volume of 50 μl by adding redistilled water and used in PCR.

PCR thermal conditions: Heating at 94° C. for 2 minutes, after which athermal cycle of 94° C. for 1 minute, 55° C. for 1 minute and 72° C. for2 minutes, repeated 30 times, followed by heating at 72° C. for 10minutes.

LM2-F3-DNA fragment coding for CDRL2, FRL₃, and a portion of CDRL₃ isprepared under the following conditions.

Composition of the Reaction Solution:

plasmid pSRPDHH DNA (European patent application EP 0 909 816 A1), 25 ng

oligonucleotide primer HKCDF22, 50 pmol

oligonucleotide primer HKCDR22, 50 pmol

dNTPs cocktail, 5 μl

10×PCR buffer, 5 μl

ampliTaq DNA polymerase, 2.5 units

The reaction solution having the above composition is adjusted to afinal volume of 50 μl by adding redistilled water and used in PCR.

PCR thermal conditions: Heating at 94° C. for 2 minutes, after which athermal cycle of 94° C. for 1 minute, 55° C. for 1 minute and 72° C. for2 minutes, repeated 30 times, followed by heating at 72° C. for 10minutes.

LM2-F4-DNA fragment coding for CDRL₃, FRL₄ and the constant region withan EcoR I restriction enzyme cleavage site added at the 3′-end isprepared under the following conditions.

Composition of the Reaction Solution:

plasmid pSRPDHH DNA, 25 ng

oligonucleotide primer HKCF12, 50 pmol

oligonucleotide primer 7ALCN, 50 pmol

dNTPs cocktail, 5 μl

10×PCR buffer, 5 μl

ampliTaq DNA polymerase, 2.5 units

The reaction solution having the above composition is adjusted to afinal volume of 50 μl by adding redistilled water and used in PCR.

PCR thermal conditions: Heating at 94° C. for 2 minutes, after which athermal cycle of 94° C. for 1 minute, 55° C. for 1 minute and 72° C. for2 minutes, repeated 30 times, followed by heating at 72° C. for 10minutes.

The amplified DNA fragments after PCR are separated by 5% polyacrylamidegel electrophoresis. The gel after electrophoresis is stained with 1μg/ml of ethidium bromide to detect the produced DNA under UV light. Therespective DNA bands thus detected are excised with a razor

b) Second Step PCR

LM2-DNA in which above described LM2-F1-DNA, LM2-F2-DNA, LM2-F3-DNA andLM2-F4-DNA fragments are fused is prepared under the followingconditions.

Composition of the Reaction Solution:

Gel fragment of LM2-F1-DNA prepared in the first step PCR,

Gel fragment of LM2-F2-DNA prepared in the first step PCR,

Gel fragment of LM2-F3-DNA prepared in the first step PCR,

Gel fragment of LM2-F4-DNA prepared in the first step PCR

oligonucleotide primer 7AL1P, 50 pmol

oligonucleotide primer 7ALCN, 50 pmol

dNTPs cocktail, 5.0 μl

10×PCR buffer, 5.0 μl

ampliTaq DNA polymerase, 2.5 units

The reaction solution having the above composition is adjusted to afinal volume of 50 μl by adding redistilled water and used in PCR.

PCR thermal conditions: Heating at 94° C. for 2 minutes, after which athermal cycle of 94° C. for 1 minute, 55° C. for 1 minute and 72° C. for2 minutes, repeated 30 times, followed by heating at 72° C. for 10minutes.

The thus prepared LM2-DNA fragment is inserted into plasmid pCR3.1DNAusing Eukaryotic TA cloning Kit (Invitrogen) following themanufacturer's protocol and introduced into the competent E. ColiTOP10F′ contained in the kit. The nucleotide sequences of these DNAsencoding the light chain of humanized TRA-8 are confirmed by the dideoxymethod (Sanger, F. S., et al., (1977), Proc. Natl. Acad. Sci. USA,74:5463-5467) using 3700 DNA Analyzer (ABI PRISM®; Perkin Elmer AppliedBiosystems, Japan).

The resulting plasmids are designated pCR3.1/M2-1 (the plasmid carryingcDNA encoding the light chain variable region of humanized TRA-8 and ahuman Ig light chain constant region).

The obtained plasmid pCR3.1/M2-1 containing LM2-DNA fragment is digestedwith the restriction enzymes Hind III and EcoR I.

One μg of cloning plasmid pHSG399 DNA is digested with the restrictionenzymes Hind III and EcoR I, and then dephosphorylated with CIP. Theresulting dephosphorylated pHSG399 DNA and LM2-DNA fragment, that havebeen digested with the restriction enzymes Hind III and EcoR I, areligated using DNA Ligation Kit Version 2.0 (Takara Syuzo, Co. Ltd.).Then, E. coli DH5α is transformed with the ligated DNA and spread ontoLB agar medium containing 0.1 mM IPTG, 0.1% X-Gal and 50 μg/mlchloramphenicol (final concentrations). The white transformants obtainedare cultured in liquid LB medium containing 50 μg/ml chloramphenicol,and plasmid DNA is extracted from the resulting culture according to thealkaline-SDS method. The extracted plasmid DNA is digested with Hind IIIand EcoR I, and then a clone carrying LM2-DNA fragment is selected by 1%agarose gel electrophoresis.

As a result of the above procedure, plasmid pHSG/M2-1-4 carrying afusion fragment of the variable region of the humanized LM2 TRA-8 lightchain and the constant region of human Igκ chain is obtained. Thetransformant E. coli strain harboring these plasmid, designated as E.coli DH5α/pHSG/M2-1-4 was deposited with International Patent OrganismDepositary. National Institute of Advanced Industrial Science andTechnology, 1-1, Higashi 1 chome Tsukuba-shi, Ibaraki-ken, 305-5466,Japan on Apr. 20, 2001, in accordance with the Budapest Treaty for theDeposit of Microorganisms, and was accorded the accession number FERMBP-7563.

3) Construction of Plasmid pSR/M2-1 (Expression Plasmid for HumanizedLM2 TRA-8 Light Chain)

The obtained plasmid pHSG/M2-1-4 carrying a fusion fragment of thevariable region of the humanized LM2 TRA-8 light chain and the constantregion of human Igκ chain is digested with the restriction enzymes HindIII and EcoR I.

One μg of cloning plasmid pSRPDHH DNA (European patent application EP0-909-816-A1) is digested with the restriction enzymes Hind III and EcoRI, and then dephosphorylated with CIP. The resulting dephospsphorylatedpSRPDHH DNA and HindIII-EcoRI DNA fragment obtained from pHSG/M2-1-4 areligated using DNA Ligation Kit Version 2.0 (Takara Syuzo, Co. Ltd.).Then, E. coli DH5α is transformed with the ligated DNA and spread ontoLB agar. The transformants obtained are cultured in liquid LB mediumcontaining 100 μg/ml ampicillin, and plasmid DNA is extracted from theresulting culture according to the alkaline-SDS method. The insertionand orientation of the desired DNA fragment in pSRPDHH vector isconfirmed by DNA sequencing using a gene sequence analyzer (ABI PRISM®3700 DNA Analyzer; Applied Biosystems).

The resulting expression plasmid carrying cDNA encoding the light chainof humanized TRA-8 is designated pSR/M2-1.

Example 20. Production of Humanized Antibody

Transfection of COS-7 cells, i.e., a cell line derived from a monkeykidney, with the expression plasmids for the humanized TRA-8 heavy chainand the humanized TRA-8 light chain obtained above is conducted byFUGENE6 transfection reagent methods (Boehringer Mannheim Biochemica)according to the instruction manual provided with the kit.

COS-7 cells (American Type Culture Collection No. CRL-1651) are grown tosemi-confluent (3×10⁶ cells/dish) in a culture dish (culture area: 57cm²: Sumitomo Bakelite) containing Dulbecco's Modified Eagle medium(hereinafter referred to as “D-MED”; Gibco BRL) supplemented with 10%fetal calf serum (hereinafter abbreviated as “FCS”; Moregate).

In the meantime, 10 μg/dish (total 5 dishes) of the humanized DR5 heavychain expression plasmid DNA (pHA15-1) and 10 μg/dish of the humanizedDR5 light chain expression plasmid DNA prepared by the alkaline-SDSmethod and cesium chloride density gradient centrifugation are mixed,and then precipitated with ethanol, followed by suspending in 5 μl/dishof dH₂O.

After 15 μl/dish of FUGENE6 Transfection regent is mixed with 180μl/dish D-MEM without FCS, this FUGENE solution (185 μl/dish) is mixedwith 5 μl/dish DNA solution containing 10 μg/dish of the humanized DR5heavy chain expression plasmid DNA and 10 μg/dish of the humanized DR5light chain expression plasmid DNA. After 15 minutes incubation at roomtemperature, the obtained plasmid suspension (200 μl) is added to thepreviously prepared COS-7 plates. After incubating in 5% CO₂ at 37° C.for 24 hours, the culture medium is changed with D-MEM without FCS.After incubating in 5% CO₂ at 37° C. for 72 hours, the culturesupernatant is recovered to purify the expression products in thesupernatant fluids. By the method as described above, COS-7 cells aretransfected with each of the following plasmid combinations:

(A): no plasmid DNA

(B): cotransfection of pHB14-1 and pSR/M2-1

The culture is then centrifuged (1,000 r.p.m., 5 minutes) and collectedthe supernatant. The supernatant is centrifuged again (9,800 r.p.m., 15minutes) and filtrated with 0.45 μm filter (ADVANTEC TOYO DISMIC-25cs,Cat #25CS045 AS). The purification of IgG from the filtrates areachieved using Protein G-POROS affinity chromatography (AppliedBiosystems) under the following conditions:

-   -   HPLC: BioCAD 700E (Applied Biosystems)    -   column: ProteinG-ID sensor cartridge (column size: 2.1 mmID×30        mm LD, bed volume: 0.1 ml: Cat #2-1002-00, Applied Biosystems)    -   elution buffer: 0.1M Glycine-HCl (pH 2.5)    -   neutralization buffer: 1M Tris-HCl (pH 8.5)    -   detection: 280 nm    -   flow rate: 1 ml/min    -   fraction size: 0.5 ml/0.5 min    -   fraction tube: 1.5 ml polypropylene microtube    -   temperature: 4° C.

After all the filtrates are applied to column, 30 ml of PBS (Sigma, Cat#1000-3) is used to wash column. When the elution buffer is applied,fraction collector started. Each fraction microtube previously contained55 μl of 1M NaCl, 110 μl of neutralization buffer and 74 μl of 2 mg/mlbovine serum albumin (Sigma, Cat #A-7030) in PBS. The fractions from No.8 through No. 10 are collected and dialyzed against 1 liter PBS (pH 7.5)at 4° C. for 1 day using Slide-A lyzer (Pierce, Cat #66450). Thedialysis buffer is changed twice.

Verification of the expression of the humanized antibodies andquantitative assay of the expression products in the culture supernatantfluids prepared is performed by ELISA with an antibody againstanti-human IgG.

To each well of a 96-well plate (MaxiSorp™, Nunc), 100 μl of goatanti-human IgG Fc specific polyclonal antibody (Kappel) dissolved at thefinal concentration of 0.5 μg/ml in adsorption buffer (0.05 M sodiumhydrogencarbonate, 0.02% sodium azide, pH 9.6) is added and the plate isincubated at 37° C. for 2 hours to cause adsorption of the antibody.Then, the plate is washed with 350 μl of PBS(−) containing 0.05%Tween®-20 (BioRad) (hereinafter referred to as “PBS-T”) five times. Tothe wells after washing, the culture supernatant diluted with D-MEMcontaining 10% FCS is added and incubated at 37° C. for 2 hours. Afterwashing again with PBS-T, 100 μl of alkaline phosphatase-labeled goatanti-human IgG Fc specific polyclonal antibody (Jackson Immuno ResearchLab.) diluted 10,000-fold with PBS-T is added to each well and incubatedat 37° C. for 2 hours. After washing again with PBS-T, a substratesolution of p-nitrophenyl phosphate obtained from Alkaline PhosphataseSubstrate kit (Bio Rad) is added according to the instruction manualprovided with the kit. After incubating at 37° C. for 0.5 to 1 hour, theabsorbance at 405 nm is measured. In the present experiments, humanplasma immunoglobulin G subclass 1 (IgG1) (Biopure AG) diluted withD-MEM containing 10%/o FCS to certain concentrations is used asconcentration reference samples of the humanized DR5 antibodiescontained in the culture supernatant fluids.

As a result, the expression and purified products in the culturesupernatant are detected specifically with the anti-human IgG antibody.The amount of human IgG antibody is 8.96 μg (800 μl).

Example 21. Apoptosis-Inducing Activity of Humanized Antibody

Jurkat cells (ATCC No. TIB-152), are used to examine theapoptosis-inducing activity of the purified humanized TRA-8 antibody.

Jurkat cells cultured in RPMI 1640 medium with 10% FCS (Gibco BRL) at37° C. for 3 days in the presence of 5% CO₂ are dispensed into each wellof a 96-well microplate (Sumitomo Bakelite) at 50 μl per well. Thehumanized TRA-8 prepared in Example 20 are adjusted to have theconcentration of the final product of interest of 100 ng/ml withRPMI1640 medium containing 10% FCS by estimating their concentrations inthe fluids according to the method described in Example 20. Each of thesolutions of the expression products thus adjusted to 100 ng/ml is usedto produce serial dilutions by repeating serial 2-fold dilution withRPMI 1640 containing 10% FCS. Each of the diluted humanized TRA-8solution is added to each well at 50 μl per well. After reacting at 37°C. for 12 hours, 50 μl of 25 μM PMS (phenazine methosulfate; SigmaChemical Co.) containing 1 mg/ml XTT(2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxyanirideinner salt; Sigma Chemical Co.) is added (final concentrations of 250μg/ml for XTT and 5 μM for PMS). After incubating for 3 hours, theabsorbance at 450 nm of each well is measured to calculate the cellviability by using the reduction ability of mitochondria as the index.

The viability of the cells in each well is calculated according to thefollowing formula:Viability (%)=100×(a−b)/(c−b)wherein “a” is the measurement of a test well, “b” is the measurement ofa well with no cells, and “c” is the measurement of a well with noantibody added.

As a result, the expression product prepared in Example 20 (humanizedTRA-8) is demonstrated to induce apoptosis in cells of T lymphoma cellline expressing human DR5 antigen.

Example 22. Reactivity of TRA-8 to Various DR5 Molecules

In order to determine the reactivity of TRA-8 to various DR5 molecules,the reactivity of TRA-8 is examined using activated lymphocytes asfollows.

First, peripheral blood samples are taken from a human (30 ml), marmoset(3 ml), and cynomolgus monkey (20 ml). The blood samples had 1 ml ofheparin (Novoheparin; Novo) added to them and the samples are thenslowly layered over an equal volume of Ficoll-Paque PLUS solution((Amersham Pharmacia Biotech.) specific gravity: 1.077 for all exceptcynomolgus monkey, which had a specific gravity of 1.072) andcentrifuged at 1,700 r.p.m. for 30 minutes in order to obtain a fractionof peripheral blood mononuclear cells. This mononuclear cell fraction iswashed twice with Hanks' balanced salt solution and then suspended inRPMI 1640 medium with 10% v/v FCS to a cell density of 1×10⁶ cells/ml.Phytohemagglutinin-P (PHA-P, Sigma Chemicals, Co.) is added to theresulting suspension to a final concentration of 5 μg/ml and the sampleincubated at 37° C. under 5% v/v CO₂ for 24 hours. After this time, thecells are recovered by centrifugation, washed and resuspended in RPMI1640 medium containing 10% v/v FCS. Then, to activate the recoveredcells, interleukin-2 (Amersham Pharmacia Biotech.) is added to thesuspension to a final concentration of 10 units/ml, and this isincubated at 37° C. under 5% v/v CO₂ for 72 hours.

An amount of the activated preparation calculated to contain 1×10⁶activated lymphocyte cells is placed in a test tube and either suspendedin 50 μl of 0.5, 1, 5, 10 μg/ml of TRA-8 in PBS or 50 μl of PBS alone.The resulting suspension is allowed to stand on ice for 1 hour, afterwhich the cells are washed 3 times with aliquots of 500 μl of PBS andthen suspended in 50 μl of 20 μg/ml FITC-labeled anti-mouse IgG antibody(Bioresource) in PBS. Using the cells suspended in 500 μl of PBS ascontrols, the fluorescence intensities are measured, using a flowcytometer (FACSCalibur; Becton Dickinson).

Distributions of cell numbers by fluorescence intensity are obtained andthe proportions of the numbers of the stained cells to those of totalcells are calculated. Further, each Kd value is calculated using theconcentration of TRA-8 and the proportions of the numbers of the stainedcells to those of total cells. Each frequency of reactivity to activatedlymphocytes of human, marmoset, and cynomologus monkey is almost same.Accordingly, TRA-8 is able to bind a wide range of primate DR5 includinghuman against which TRA-8 is originally prepared.

Example 23. Escalating Dose Study of TRA-8 in Marmosets

An escalating dose preliminary toxicity study of TRA-8 is performedusing 1 male and 1 female marmoset. Three sets of single intravenousdosing, which are separated by a 7-day withdrawal period, are carriedout. The dose of TRA-8 is set at 50, 250 and 1250 μg/body. Forty-eighthours after each treatment, blood is collected from the femoral vein andthe plasma is prepared. Plasma aspartate aminotransferase and alanineaminotransferase activities are measured using an analyzer (FUJIDRI-CHEM: Fuji Film Medical Co., Ltd.). All blood is taken without anyanesthetization. As a result, no evidences indicating hepatic injury arenoted in plasma biochemical examination after each treatment.

Example 24. In Vitro and In Vivo Pharmacological Studies of TRA-8Against Cancer Cells

In order to determine whether TRA-8 has the therapeutic efficacy inanti-cancer therapy, in vitro killing activity of TRA-8 using variouscancer cell lines is examined as follows.

Various cancer cells (2-8×10³ cells/50 μl) cultured in RPMI 1640 medium(for Jurkat), DMEM medium (for HCT-116), MEM-R (for WiDr), or DMEM-F12(for COL2-Jck) obtained from Gibco BRL with 10% FCS (Gibco BRL) at 37°C. in the presence of 5% CO₂ are dispensed into each well of a 96-wellmicroplate (Sumitomo Bakelite). TRA-8 are adjusted to have theconcentration of the final product of interest of 100 ng/ml with mediumcontaining 10% FCS. The TRA-8 solution (100 ng/ml) is used to produceserial dilutions by repeating serial 2-fold dilution with mediumcontaining 10% FCS. Each of the diluted TRA-8 solution is added to eachwell at 50 μl per well and incubated at 37° C. After reacting at 37° C.for 72 hours, 50 μl of 25 μM PMS (phenazine methosulfate; Sigma ChemicalCo.) containing 1 mg/ml XTT is added (final concentrations of 250 μg/mlfor XTT and 5 μM for PMS). After incubating for 3 hours, the absorbanceat 450 nm of each well is measured to calculate the cell viability byusing the reduction ability of mitochondria as the index.

The viability of the cells in each well is calculated according to thefollowing formula:Viability (%)=100×(a−b)/(c−b)wherein “a” is the measurement of a test well, “b” is the measurement ofa well with no cells, and “c” is the measurement of a well with noantibody added.

The results are shown in Table 3, below.

TABLE 3 ED50 Cells (μg/ml) Jurkat 0.001-0.01 HCT-116 0.004-0.02 WiDr0.007-0.03 COL2-Jck 2.28

Various cancer cell lines are strongly induced apoptosis by TRA-8 underthe in vitro conditions.

Furthermore, the in vivo anti-tumor effect of TRA-8 in nude micetransplanted with WiDr cells is determined, because TRA-8 is notcross-reactive with murine DR5.

TRA-8 anti-human DR5 antibody is administered to nude mice bearing humanxenografts that express the human DR5 molecule. The mice used were 6week-old BALb/c nude/nude mice (female, from Clea Japan Inc.), whichwere transplanted with human colon cancer cell lines WiDr (5 mm). At oneday after tumor transplantation, these transplanted mice are dailytreated with the intra-articular injection of TRA-8 (5 μg/body) to 14times. WiDr tumor growth is daily determined by the size of the tumormass. The results are shown in Table 4, below.

TABLE 4 8 days 11 days 15 days 18 days 22 days 25 days Control (PBS) 196± 249 ± 469 ± 584 ± 833 ± 1193 ± SD 55 77 149 230 274 419 TRA-8 158 ± 97± 155 ± 195 ± 365 ± 530 ± SD 78 30 60 58 91 135

In this model, while all untreated animals exhibited visible tumorgrowth, tumor growth in TRA-8 treated animals is inhibited asdemonstrated by the size of tumor. This result indicated that TRA-8 iseffective in the elimination of tumor cells in vivo.

Example 25. Combination Study of TRA-8

Human prostate cancer cell line PC-3 is obtained from American TissueCulture Collection (ATCC) and maintained in F-12K Nutrient Mixture(21127-022, Gibco BRL) containing 10% fetal bovine serum (FBS, Hyclone),1% L-Glutamine-200 mM (25030-149, Gibco BRL) and 0.5% PenicillinStreptomycin Solution (P-7539, Sigma). RPMI1640 medium (MED-008, IWAKI)supplemented with 10% FBS and 0.5% Penicillin Streptomycin Solution isused in the following experiment. Exponentially growing PC-3 cells arecollected by trypsinization and washed twice with fresh medium. Thecells are then counted, resuspended in fresh medium at a density of5×10⁴ cells/ml and distributed in triplicate into flat-bottomed 96 wellplates (3598, Corning-Coster) in a total volume of 100 μL/well one daybefore the start of the experiment. A representative anti-cancer drug,Paclitaxcel (169-18611, Wako) dissolved in dimethylsulfoxide (10 mg/ml)is diluted in fresh medium and then added to the 96-well platescontaining the cells at 50 μl/well. The final concentrations ofdimethylsulfoxide are less than 0.1%. After incubation for 24 hr at 37°C. in 5% CO₂ atmosphere, TRA-8 diluted in fresh medium is added to thewells. After incubation for a further 24 hr, 50 μl of Minimum EssentialMedium (11095-098, Gibco BRL) containing 1 mg/ml of XTT and 25 mM of PMSis added to the wells and the plates are incubated for 6 hr. OD450 isthen measured by SPECTRA MAX 250 (Molecular Devices) and the cellviability is calculated as follows.Cell viability (%)=(OD450 for the well containing cells treated withTaxol and/or TRA-8 (agent(s))−OD450 for the well containing neithercells nor agent)×100/(OD450 for the well containing cells with noagent−OD450 for the well containing neither cells nor agent)

The result of the above assay for TRA-8 combined with a representativeanti-cancer drug, Paclitaxel, is followed. Paclitaxel reduced the cellviability of PC-3 cells but more than 40% of the signals indicatingviable cancer cells still remained at concentrations of up to 200 nM.Notably, the addition of 0.1 ng/ml of TRA-8 greatly decreased the cellviability of the cancer cells, up to 10%, even though no reduction incell viability is seen after a single application of TRA-8 at thisconcentration. This result clearly indicates that TRA-8 exhibitedanti-cancer activity synergistically when combined with otheranti-cancer drugs.

Example 26. Analysis of Other Type Humanized Antibodies of TRA-8

(1) Designing Humanized Antibodies

Construction of a humanized version of TRA-8 is performed by the methodgenerally known as CDR grafting, mAB58′CL antibody is used as anacceptor as described in Reference Example 2 and the CDR regions ofTRA-8 antibody is grafted on the acceptor. In the framework region, someamino acids are grafted on the acceptor from either TRA-8 or humanconsensus sequences by the criteria given by Queen et al. (Proc. Natl.Acad. Sci. USA 86, 10029-10033, (1989)) and humanized TRA-8 sequencesare constructed as described hereinbelow.

(2) Construction of Plasmid Carrying the Heavy Chain Variable Region DNAof Other Types Humanized or Mouse TRA-8

As shown in SEQ ID No. 56 of the Sequence Listing, H1 type-humanizationof the amino acid sequences of the heavy chain of the mouse anti-humanDR5 antibody TRA-8 entailed replacing the 3rd amino acid (methionine),the 13th amino acid (lysine), the 19th amino acid (lysine), the 40thamino acid (threonine), the 42nd amino acid (glutamic acid), the 44thamino acid (arginine), the 84th amino acid (serine), the 88th amino acid(serine), the 93rd amino acid (methionine), the 114th amino acid(threonine), the 115th amino acid (leucine) with glutamine, glutamine,arginine, alanine, glycine, glycine, asparagine, alanine, valine,leucine, and valine, respectively.

As shown in SEQ ID No. 59 of the Sequence Listing, H3 type-humanizationof the amino acid sequences of the heavy chain of the mouse anti-humanDR5 antibody TRA-8 entailed replacing the 13th amino acid (lysine), the19th amino acid (lysine), the 40th amino acid (threonine), the 42ndamino acid (glutamic acid), the 44th amino acid (arginine), the 88thamino acid (serine), the 93rd amino acid (methionine), the 114th aminoacid (threonine), the 115th amino acid (leucine) with glutamine,arginine, alanine, glycine, glycine, alanine, valine, leucine, andvaline, respectively.

As shown in SEQ ID No. 60 of the Sequence Listing, H4 type-humanizationof the amino acid sequences of the heavy chain of the mouse anti-humanDR5 antibody TRA-8 entailed replacing the 13th amino acid (lysine), the19th amino acid (lysine), the 88th amino acid (serine), the 93rd aminoacid (methionine), the 114th amino acid (threonine), the 115th aminoacid (leucine) with glutamine, arginine, alanine, valine, leucine, andvaline, respectively.

As shown in SEQ ID No. 61 of the Sequence Listing, the plasmid carryingthe heavy chain variable region DNA of chimeric TRA-8 is designated as“M type”. In addition, humanized TRA-8 described in Example 17 and 18 isdesignated as “H2 type”.

The plasmids carrying DNA encoding heavy chain variable region ofhumanized or chimeric TRA-8 are constructed as follows.

PCR is used to construct the following DNA sequences, each of whichcomprised described above:

The following 24 oligonucleotide are synthesized:

(A; SEQ ID No. 32) 5′- ttggataagc ttggcttgac ctcaccatgg gatggagctgtatcatcctc ttcttggtag caacagctac aggtgtccac -3′; (B; SEQ ID No. 33)5′- tctgaagtaa tgctggtgga gtctggggga ggcttagtacagcctggagg gtccctgaga ctctcctgtg cagcctctgg -3′; (B2; SEQ ID No. 57)5′- tctgaagtac agctggtgga gtctggggga ggcttagtacagcctggagg gtccctgaga ctctcctgtg cagcctctgg -3′; (B3; SEQ ID No. 66)5′- tctgaagtaa tgctggtgga gtctggggga ggcttagtaaagcctggagg gtccctgaaa ctctcctgtg cagcctctgg -3′; (C; SEQ ID No. 34)5′- attcactttc agtagttatg taatgtcttg ggttcggcaggcaccaggga agggtctgga gtgggttgca accattagta -3′; (C2; SEQ ID No. 64)5′- attcactttc agtagttatg taatgtcttg ggttcggcagactccagaga agaggctgga gtgggttgca accattagta -3′; (D; SEQ ID No 35)5′- gtggtggtag ttacacctac tatccagaca gtgtgaagggccgattcacc atctccagag acaatgccaa gaacaccctg -3′; (E; SEQ ID No. 36)5′- tatctgcaaa tgaacagtct gagagcagag gacacggctgtttattactg tgcaagaagg ggtgactcta tgattacgac -3′; (E2; SEQ ID No. 62)5′- tatctgcaaa tgagcagtct gagagcagag gacacggctgtttattactg tgcaagaagg ggtgactcta tgattacgac -3′; (E3; SEQ ID No. 67)5′- tatctgcaaa tgagcagtct gagatctgag gacacggctatgtattactg tgcaagaagg ggtgactcta tgattacgac -3′; (F; SEQ ID No. 37)5′- ggactactgg ggccaaggga ccctggtcac agtctcctca gcctccacc aagggcccat cggtc -3′; (F2; SEQ ID No. 68)5′- ggactactgg ggccaaggga ccactctcac agtctcctca gcctccacc aagggcccat cggtc -3′; (G; SEQ ID No. 38)5′- ctaccaagaa gaggatgata cagctccatc ccatggtgag gtcaagccaa gcttatccaa -3′; (H; SEQ ID No. 39)5′- tctcagggac cctccaggct gtactaagcc tcccccagac tccaccagca ttacttcaga gtggacacct gtagctgttg -3′; (H2; SEQ ID No. 58)5′- tctcagggac cctccaggct gtactaagcc tcccccagac tccaccagct gtacttcaga gtggacacct gtagctgttg -3′; (H3; SEQ ID No. 69)5′- tttcagggac cctccaggct ttactaagcc tcccccagac tccaccagca ttacttcaga gtggacacct gtagctgttg -3′; (I; SEQ ID No. 40)5′- tccagaccct tccctggtgc ctgccgaacc caagacatta cataactact gaaagtgaat ccagaggctg cacaggagag -3′; (I2; SEQ ID No. 65)5′- tccagcctct tctctggagt ctgccgaacc caagacatta cataactact gaaagtgaat ccagaggctg cacaggagag -3′; (J; SEQ ID No. 41)5′- ctctggagat ggtgaatcgg cccttcacac tgtctggata gtaggtgtaa ctaccaccac tactaatggt tgcaacccac -3′; (K; SEQ ID No. 42)5′- ccttcttgca cagtaataaa cagccgtgtc ctctgctctc agactgttca tttgcagata cagggtgttc ttggcattgt -3′; (K2; SEQ ID No. 63)5′- ccttcttgca cagtaataaa cagccgtgtc ctctgctctc agactgttca tttgcagata cagggtgttc ttggcattgt -3′; (K3; SEQ ID No. 70)5′- ccttcttgca cagtaataca tagccgtgtc ctcagatctc agactgctca tttgcagata cagggtgttc ttggcattgt -3′; (L; SEQ ID No. 43)5′- gaccgatggg cccttggtgg aggctgagga gactgtgacc agggtccctt ggccccagta gtccgtcgta atcatagagt  cacc -3′ and(L2; SEQ ID No. 71) 5′- gaccgatggg cccttggtgg aggctgagga gactgtgaga gtggtccctt ggccccagta gtccgtcgta atcatagagt cacc -3′.The following 2 PCR primers are synthesized as described above:(P1; SEQ ID No. 44) 5′- ttggataagc ttggcttgac -3′; and(P2; SEQ ID No. 45) 5′- gaccgatggg cccttggtgg a -3′.

The synthesis of H1 type DNA encoding a polypeptide chain comprising asecretion signal sequence, a variable region of humanized TRA-8 heavychain and the 8 amino acid residues at the N-terminus of the IgG-CH1region is performed using a combination of PCR respectively.

The H1 type-DNA fragment is prepared as follows.

Composition of the PCR Reaction Solution:

oligonucleotide A, 10 pmol;

oligonucleotide B2, 10 pmol;

oligonucleotide C, 10 pmol;

oligonucleotide D, 10 pmol;

oligonucleotide E, 10 pmol;

oligonucleotide F, 10 pmol;

oligonucleotide G, 10 pmol;

oligonucleotide H2, 10 pmol;

oligonucleotide I, 10 pmol;

oligonucleotide J, 10 pmol;

oligonucleotide K, 10 pmol;

oligonucleotide L, 10 pmol;

oligonucleotide primer P1, 2 μM;

oligonucleotide primer P2, 2 μM;

10× Pyrobest™ buffer II, 10 μl;

dNTP mix, 8 μl;

Pyrobest™ DNA polymerase, 0.5 μl; and

Redistilled water to a final volume of 50 μl.

The PCR reaction is conducted as follows. The solution is first heatedat 94° C. for 5 minutes, after which a cycle of heating to 98° C. for 10second, 55′C for 30 second and 72° C. for 1 minute, is repeated 7 times.After completion of this procedure, the reaction solution is heated at72° C. for 15 minutes.

After phenol extraction and ethanol precipitation, the resulting DNAprecipitate is vacuum-dried, dissolved in a minimum of redistilledwater, and separated by 3% agarose gel electrophoresis. Afterelectrophoresis, the gel is stained with a 1 μg/ml aqueous solution ofethidium bromide to allow detection of DNA under UV light. The DNA bandscorresponding to H1 type-DNA is cut out using a razor blade and elutedfrom the gel using Geneclean® Spin Kit (BIO 101, CA, USA). After phenolextraction, the eluted DNA is then concentrated by centrifugation at7,500×g, followed by ethanol precipitation, and finally dissolved in 5μl of distilled water.

The synthesis of H3 type DNA encoding a polypeptide chain comprising asecretion signal sequence, a variable region of humanized TRA-8 heavychain and the 8 amino acid residues at the N-terminus of the IgG-CH1region is performed using a combination of PCR respectively.

The H3 type-DNA fragment is prepared as follows.

Composition of the PCR Reaction Solution:

oligonucleotide A, 10 pmol;

oligonucleotide B, 10 pmol;

oligonucleotide C, 10 pmol;

oligonucleotide D, 10 pmol;

oligonucleotide E2, 10 pmol;

oligonucleotide F, 10 pmol;

oligonucleotide G, 10 pmol;

oligonucleotide H, 10 pmol;

oligonucleotide I, 10 pmol;

oligonucleotide J, 10 pmol;

oligonucleotide K2, 10 pmol;

oligonucleotide L, 10 pmol;

oligonucleotide primer P1, 2 μM;

oligonucleotide primer P2, 2 μM;

10× Pyrobest™ buffer II, 10 μl;

dNTP mix, 8 μl;

Pyrobest™ DNA polymerase, 0.5 μl; and

Redistilled water to a final volume of 50 μl.

The PCR reaction is conducted as follows. The solution is first heatedat 94° C. for 5 minutes, after which a cycle of heating to 98° C. for 10second, 55° C. for 30 second and 72° C. for 1 minute, is repeated 7times. After completion of this procedure, the reaction solution isheated at 72° C. for 15 minutes.

After phenol extraction and ethanol precipitation, the resulting DNAprecipitate is vacuum-dried, dissolved in a minimum of redistilledwater, and separated by 3% agarose gel electrophoresis. Afterelectrophoresis, the gel is stained with a 1 μg/ml aqueous solution ofethidium bromide to allow detection of DNA under UV light. The DNA bandscorresponding to H3 type-DNA is cut out using a razor blade and elutedfrom the gel using Geneclean® Spin Kit. After phenol extraction, theeluted DNA is then concentrated by centrifugation at 7,500×g, followedby ethanol precipitation, and finally dissolved in 5 μl of distilledwater.

The synthesis of H4 type DNA encoding a polypeptide chain comprising asecretion signal sequence, a variable region of humanized TRA-8 heavychain and the 8 amino acid residues at the N-terminus of the IgG-CH1region is performed using a combination of PCR respectively.

The H4 type-DNA fragment is prepared as follows.

Composition of the PCR Reaction Solution:

oligonucleotide A, 10 pmol;

oligonucleotide B, 10 pmol;

oligonucleotide C2, 10 pmol;

oligonucleotide D, 10 pmol;

oligonucleotide E2, 10 pmol;

oligonucleotide F, 10 pmol;

oligonucleotide G, 10 pmol;

oligonucleotide H, 10 pmol;

oligonucleotide I2, 10 pmol;

oligonucleotide J, 10 pmol;

oligonucleotide K2, 10 pmol;

oligonucleotide L, 10 pmol;

oligonucleotide primer P1, 2 μM;

oligonucleotide primer P2, 2 μM;

10× Pyrobest™ buffer II, 10 μl;

dNTP mix, 8 μl;

Pyrobest™ DNA polymerase, 0.5 μl; and

Redistilled water to a final volume of 50 μl.

The PCR reaction is conducted as follows. The solution is first heatedat 94° C. for 5 minutes, after which a cycle of heating to 98° C. for 10second, 55° C. for 30 second and 72° C. for 1 minute, is repeated 7times. After completion of this procedure, the reaction solution isheated at 72° C. for 15 minutes.

After phenol extraction and ethanol precipitation, the resulting DNAprecipitate is vacuum-dried, dissolved in a minimum of redistilledwater, and separated by 3% agarose gel electrophoresis. Afterelectrophoresis, the gel is stained with a 1 μg/ml aqueous solution ofethidium bromide to allow detection of DNA under UV light. The DNA bandscorresponding to H4 type-DNA is cut out using a razor blade and elutedfrom the gel using Geneclean® Spin Kit. After phenol extraction, theeluted DNA is then concentrated by centrifugation at 7,500×g, followedby ethanol precipitation, and finally dissolved in 5 μl of distilledwater.

The synthesis of M type DNA encoding a polypeptide chain comprising asecretion signal sequence, a variable region of chimeric TRA-8 heavychain and the 8 amino acid residues at the N-terminus of the IgG-CH1region is performed using a combination of PCR respectively.

The M type-DNA fragment is prepared as follows.

Composition of the PCR Reaction Solution:

oligonucleotide A, 10 pmol;

oligonucleotide B3, 10 pmol;

oligonucleotide C2, 10 pmol;

oligonucleotide D, 10 pmol;

oligonucleotide E3, 10 pmol;

oligonucleotide F2, 10 pmol;

oligonucleotide G, 10 pmol;

oligonucleotide H3, 10 pmol;

oligonucleotide I2, 10 pmol;

oligonucleotide J, 10 pmol;

oligonucleotide K3, 10 pmol;

oligonucleotide L2, 10 pmol;

oligonucleotide primer P1, 2 μM;

oligonucleotide primer P2, 2 μM;

10× Pyrobest™ buffer II, 10 μl;

dNTP mix, 8 μl;

Pyrobest™ DNA polymerase, 0.5 μl; and

Redistilled water to a final volume of 50 μl.

The PCR reaction is conducted as follows. The solution is first heatedat 94° C. for 5 minutes, after which a cycle of heating to 98° C. for 10second, 55° C. for 30 second and 72° C. for 1 minute, is repeated 7times. After completion of this procedure, the reaction solution isheated at 72° C. for 15 minutes.

After phenol extraction and ethanol precipitation, the resulting DNAprecipitate is vacuum-dried, dissolved in a minimum of redistilledwater, and separated by 3% agarose gel electrophoresis. Afterelectrophoresis, the gel is stained with a 1 μg/ml aqueous solution ofethidium bromide to allow detection of DNA under UV light. The DNA bandscorresponding to M type-DNA is cut out using a razor blade and elutedfrom the gel using Geneclean® Spin Kit. After phenol extraction, theeluted DNA is then concentrated by centrifugation at 7,500×g, followedby ethanol precipitation, and finally dissolved in 5 μl of distilledwater.

The resulting, each extracted DNA (H1 type, H3 type, H4 type, and Mtype) is cloned using pGEM-T Easy vector (Promega) as follows:

-   -   The DNA fragment recovered from the PCR reaction (H1, H3, H4 or        M), 5 μl;    -   10× Taq polymerase buffer, 1 μl;    -   dNTP mixture, 1 μl    -   Taq polymerase (5 unit/ml), 1 μl; and    -   redistilled water to a final volume of 10 μl.

After the above each solution is reacted at 70° C. for 30 minutes, eachDNA solution and pGEM-T Easy vector are ligated using a DNA Ligation KitVersion 2.0 (Takara Shuzo Co., Ltd.) using the manufacturer's protocol.

After 4 hours incubation at 15° C., 2 μl of the incubated reactionsolution is mixed with 100 μl of competent E. coli strain JM109 at acell density of 1-2×10⁹ cells/ml (Takara Shuzo Co., Ltd.), and themixture is kept on ice for 30 minutes, then at 42° C. for 30 seconds,and again on ice for 1 minute. Then, 500 μl of SOC medium (2% v/vtryptone, 0.5% w/v yeast extract, 0.05% w/v sodium chloride, 2.5 mM w/vpotassium chloride, 1 mM magnesium chloride, and 20 mM glucose) is addedthe mixture, which is incubated for a further hour, with shaking.Transformant strains are then isolated, and plasmid DNA is prepared fromthe strains as described in “Molecular Cloning: A Laboratory Manual”.The nucleotide sequence of this DNA encoding the heavy chain ofhumanized or mouse TRA-8 is confirmed by the dideoxy method,respectively (Sanger, F. S., et al., (1977), Proc. Natl. Acad. Sci. USA,74:5463-5467) using 3700 DNA. Analyzer (ABI PRISM®; Perkin Elmer AppliedBiosystems, Japan).

The resulting plasmid is designated pHA15 (the plasmid carrying cDNAencoding the H1-type heavy chain of humanized TRA-8), pHC10 (the plasmidcarrying cDNA encoding the H3-type heavy chain of humanized TRA-8),pHD21 (the plasmid carrying cDNA encoding the H4-type heavy chain ofhumanized TRA-8), and pM11 (the plasmid carrying cDNA encoding the heavychain of chimeric TRA-8). The transformant E. coli strains harboringthese plasmid, designated as E. coli JM109/pHA15. E. coli JM109/pHC10,E. coli JM109/pHD21 and E. coli JM109/pM11 were deposited withInternational Patent Organism Depositary, National Institute of AdvancedIndustrial Science and Technology, 1-1, Higashi 1 chome Tsukuba-shi,Ibaraki-ken, 305-5466, Japan on Apr. 20, 2001, in accordance with theBudapest Treaty for the Deposit of Microorganisms, and was accorded theaccession number FERM BP-7555, FERM BP-7557, FERM BP-7558, and FERMBP-7559, respectively.

(3) Construction of Expression Plasmids Carrying the Heavy ChainVariable Region DNA of Several Types Humanized or Mouse TRA-8

Recombinant expression vector for animal cells are constructed byinserting the DNA encoding the heavy chain of H1 type, H3 type, and H4type humanized or M type chimeric TRA-8 (cloned in above) as follows.

One μg of plasmid pSRHHH3 (European patent application EP 0 909 816 A1)carrying the heavy chain variable region of humanized anti-Fasmonoclonal antibody HFE7A and human IgG1 constant region genomic DNA, anexpression vector for mammalian cells, is digested with the restrictionenzymes HindIII and ApaI, and separated by 3% agarose gelelectrophoresis. After electrophoresis, the gel is stained with a 1μg/ml aqueous solution of ethidium bromide to allow detection of DNAunder UV light. The vector DNA bands containing human IgG1 constantregion genomic DNA without the heavy chain variable region of humanizedHFE7A are cut out using a razor blade and eluted from the gel usingGeneclean® Spin Kit. After phenol extraction, the eluted DNA is thenconcentrated by centrifugation at 7,500×g, followed by ethanolprecipitation, and finally dissolved in 5 μl of distilled water and thendephosphorylated using CIP. The resulting digested, dephosphorylatedplasmid (100 ng) is ligated with 1 μg of the DNA fragment of pHA15,pHC10, pHD21, or pM11 containing the DNA encoding the heavy chainvariable region of humanized or chimeric TRA-8, which had also beendigested with HindIII and ApaI, using a DNA Ligation Kit Version 2.0(Takara Shuzo Co., Ltd.). The ligation mixture is then used to transformE. coli JM109, which is then plated on LB agar plates containing 50μg/ml ampicillin.

The transformants obtained by this method are cultured in 2 ml of liquidLB medium containing 50 μg/ml ampicillin at 37° C. overnight, andplasmid DNA is subsequently extracted from the resulting culture by thealkaline-SDS method.

The extracted plasmid DNA is digested with HindIII and ApaI, andsubjected to 3% w/v agarose gel electrophoresis to confirm the presenceor absence of the insert of the DNA encoding the heavy chain variableregion of humanized or chimeric TRA-8. The insertion and orientation ofthe desired DNA fragment in the vector is confirmed by DNA sequencingusing a gene sequence analyzer (ABI PRISM®) 3700 DNA Analyzer; AppliedBiosystems). The resulting expression plasmids carrying cDNA encodingthe heavy chain of humanized or chimeric TRA-8 were designated pHA15-1,pHC10-3, pHD21-1, and pM11-1, respectively.

(4) Construction of Vectors for the Humanized Light Chains

(4.1) Construction of an Expression Vector for the Light Chain of theHumanized Antibody (LM1 Type)

As shown in SEQ ID No. 72 of the Sequence Listing, other humanization(LM1 type) of the amino acid sequences of the light chain of the mouseanti-human DR5 antibody TRA-8 entailed replacing the 3rd amino acid(valine), 8th amino acid (histidine), 9th amino acid (lysine), 10thamino acid (phenylalanine), 11th amino acid (methionine), 13th aminoacid (threonine), 20th amino acid (serine), 42nd amino acid (glutamine),43rd (serine), 60th amino acid (aspartic acid), 63rd amino acid(threonine), 77th amino acid (asparagine), 78th amino acid (valine),80th amino acid (serine) 83rd amino acid (leucine), 85th amino acid(aspartic acid), 87th amino acid (phenylalanine), and 99th amino acid(glycine) 103rd amino acid (leucine) and 108th amino acid (alanine) fromthe N-terminus of the amino acid sequence of the TRA-8 light chain arereplaced with glutamine, proline, serine, serine, leucine, alanine,threonine, lysine, alanine, serine, serine, serine, leucine, proline,phenylalanine, threonine, tyrosine, glutamine, valine and threoninerespectively. The resulting sequence is designated LM1.

Expression plasmids carrying this type of humanized light chain aminoacid sequences of the anti-human DR5 antibody TRA-8 (LM1 type, SEQ IDNo. 72 of the Sequence Listing) are constructed as follows.

1) Synthesis of Primers for Preparing the Variable and Constant Regionsof the Light Chain of Humanized TRA-8 (LM1 Type)

DNA coding for the LM1 polypeptide chain (SEQ ID No. 72 of the SequenceListing), each of which is a fusion of the variable region of humanizedanti-DR5 antibody TRA-8 light chain (LM1 type) and the constant regionof the human Ig light chain (κ chain), are respectively synthesized byusing combinations of PCR.

Further to 7AL1P (SEQ ID No. 47), 7ALCN (SEQ ID No. 48), HKCDF11 (SEQ IDNo. 50), HKCDR12 (SEQ ID No. 51), HKCDF22 (SEQ ID No. 52), HKCDR22 (SEQID No. 53), and HKCF12 (SEQ ID No. 54).

The following oligonucleotide primers are synthesized for PCR:

-   -   5′-gtcccccaca gatgcagaca aagaacttgg agattgggtc atctgaatgt        caccagtgga-3′ (FIKSPR12; SEQ ID No. 77).        2) Construction of Plasmid pCR3.1/LM1-2 (Cloning of Humanized        TRA-8 Light Chain Type LM1)

LM1-DNA fragment coding for the amino acid sequence as defined in SEQ IDNo. 72 of the same is prepared by performing 2-step PCR, inserted into aplasmid vector and cloned in E. coli.

a) First Step PCR

LM1-F1-DNA fragment coding for a secretion signal sequence and a portionof FRL₁ region with a Hind III restriction enzyme cleavage site added atthe 5′-end is prepared under the following conditions. The templateplasmids, pHSGHM17 and pSRPDHH, are obtained by following thedescription in a European patent application EP 0 909 816 A1.

Composition of the Reaction Solution:

plasmid pHSGHM17 DNA, 25 ng

oligonucleotide primer 7AL1P, 50 pmol

oligonucleotide primer HKSPR12, 50 pmol

dNTPs cocktail, 5 μl

10×PCR buffer, 5 μl

ampliTaq DNA polymerase (PerkinElmer), 2.5 units

The reaction solution having the above composition is adjusted to afinal volume of 50 μl by adding redistilled water and used in PCR.

PCR thermal conditions: Heating at 94° C. for 2 minutes, after which athermal cycle of 94° C. for 1 minute, 55° C. for 1 minute and 72° C. for2 minutes, repeated 30 times, followed by heating at 72° C. for 10minutes.

LM1-F2-DNA fragment coding for a portion of FRL₁, CDRL₁, FRL₂, and CDRL₂is prepared under the following conditions.

Composition of the Reaction Solution:

plasmid pL28 DNA, 25 ng

oligonucleotide primer HKCDF11, 50 pmol

oligonucleotide primer HKCDR12, 50 pmol

dNTPs cocktail, 5 μl

10×PCR buffer, 5 μl

ampliTaq DNA polymerase, 2.5 units

The reaction solution having the above composition is adjusted to afinal volume of 50 μl by adding redistilled water and used in PCR.

PCR thermal conditions: Heating at 94° C. for 2 minutes, after which athermal cycle of 94° C. for 1 minute, 55° C. for 1 minute and 72° C. for2 minutes, repeated 30 times, followed by heating at 72° C. for 10minutes.

LM1-F3-DNA fragment coding for CDRL2, FRL₃, and a portion of CDRL₃ isprepared under the following conditions.

Composition of the Reaction Solution:

plasmid pSRPDHH DNA, 25 ng

oligonucleotide primer HKCDF22, 50 pmol

oligonucleotide primer HKCDR22, 50 pmol

dNTPs cocktail, 5 μl

10×PCR buffer, 5 μl

ampliTaq DNA polymerase, 2.5 units

The reaction solution having the above composition is adjusted to afinal volume of 50 μl by adding redistilled water and used in PCR.

PCR thermal conditions: Heating at 94° C. for 2 minutes, after which athermal cycle of 94° C. for 1 minute, 55° C. for 1 minute and 72° C. for2 minutes, repeated 30 times, followed by heating at 72° C. for 10minutes.

LM1-F4-DNA fragment coding for CDRL₃, FRL₄ and the constant region withan EcoR I restriction enzyme cleavage site added at the 3′-end isprepared under the following conditions.

Composition of the Reaction Solution:

plasmid pSRPDHH DNA, 25 ng

oligonucleotide primer HKCF12, 50 pmol

oligonucleotide primer 7ALCN, 50 pmol

dNTPs cocktail, 5 μl

10×PCR buffer, 5 μl

ampliTaq DNA polymerase, 2.5 units

The reaction solution having the above composition is adjusted to afinal volume of 50 μl by adding redistilled water and used in PCR.

PCR thermal conditions: Heating at 94° C. for 2 minutes, after which athermal cycle of 94° C. for 1 minute, 55° C. for 1 minute and 72° C. for2 minutes, repeated 30 times, followed by heating at 72*C for 10minutes.

The amplified DNA fragments after PCR are separated by 5% polyacrylamidegel electrophoresis. The gel after electrophoresis is stained with 1μg/ml of ethidium bromide to detect the produced DNA under UV light. Therespective DNA bands thus detected are excised with a razor

b) Second Step PCR

LM1-DNA in which above described LM1-F1-DNA, LM1-F2-DNA, LM1-F3-DNA andLM1-F4-DNA fragments are fused is prepared under the followingconditions.

Composition of the Reaction Solution:

Gel fragment of LM1-F1-DNA prepared in the first step PCR,

Gel fragment of LM1-F2-DNA prepared in the first step PCR,

Gel fragment of LM1-F3-DNA prepared in the first step PCR,

Gel fragment of LM1-F4-DNA prepared in the first step PCR

oligonucleotide primer 7AL1P, 50 pmol

oligonucleotide primer 7ALCN, 50 pmol

dNTPs cocktail, 5.0 μl

10×PCR buffer, 5.0 μl

ampliTaq DNA polymerase, 2.5 units

The reaction solution having the above composition is adjusted to afinal volume of 50 μl by adding redistilled water and used in PCR.

PCR thermal conditions: Heating at 94° C. for 2 minutes, after which athermal cycle of 94° C. for 1 minute, 55° C. for 1 minute and 72° C. for2 minutes, repeated 30 times, followed by heating at 72° C. for 10minutes.

The thus prepared LM1-DNA fragment is inserted into plasmid pCR3.1DNAusing Eukaryotic TA cloning Kit (InVitrogen) following themanufacturer's protocol and introduced into the competent E. ColiTOP10F′ contained in the kit. The nucleotide sequences of these DNAsencoding the light chain of humanized TRA-8 (LM1 type) are confirmed bythe dideoxy method (Sanger, F. S., et al., (1977), Proc. Natl. Acad.Sci. USA, 74:5463-5467) using 3700 DNA Analyzer (ABI PRISM®, PerkinElmer Applied Biosystems, Japan).

The resulting plasmids are designated pCR3.1/LM1-2 (the plasmid carryingcDNA encoding the light chain variable region of humanized TRA-8 (LM1type) and a human Ig light chain constant region).

The obtained plasmid pCR3.1/LM1-2 containing LM1-DNA fragment isdigested with the restriction enzymes Hind III and EcoR I.

One μg of cloning plasmid pHSG399 DNA is digested with the restrictionenzymes Hind III and EcoR I, and then dephosphorylated with CIP. Theresulting dephosphorylated pHSG399 DNA and LM1-DNA fragment, that hadbeen digested with the restriction enzymes Hind III and EcoR I, areligated using DNA Ligation Kit Version 2.0 (Takara Syuzo, Co. Ltd.).Then, E. coli DH5α is transformed with the ligated DNA and spread ontoLB agar medium containing 0.1 mM IPTG, 0.1% X-Gal and 50 μg/mlchloramphenicol (final concentrations). The white transformants obtainedare cultured in liquid LB medium containing 50 μg/ml chloramphenicol,and plasmid DNA is extracted from the resulting culture according to thealkaline-SDS method. The extracted plasmid DNA is digested with Hind IIIand EcoR I, and then a clone carrying LM1-DNA fragment is selected by 1%agarose gel electrophoresis.

As a result of the above procedure, plasmid pHSG/M1-2-2 carrying afusion fragment of the variable region of the humanized LM1 TRA-8 lightchain and the constant region of human Igκ chain is obtained. Thetransformant E. coli strain harboring these plasmid, designated as E.coli DH5/pHSG/M1-2-2 was deposited with International Patent OrganismDepositary, National institute of Advanced Industrial Science andTechnology, 1-1, Higashi 1 chome Tsukuba-shi, Ibaraki-ken, 305-5466,Japan on Apr. 20, 2001, in accordance with the Budapest Treaty fir theDeposit of Microorganisms, and was accorded the accession number FERMBP-7562.

3) Construction of Plasmid pSR/LM1-2 (Expression Plasmid for HumanizedLM1 TRA-8 Light Chain)

The obtained plasmid pHSG/M1-2-carrying a fusion fragment of thevariable region of the humanized LM1 TRA-8 light chain and the constantregion of human Igκ chain is digested with the restriction enzymes HindIII and EcoR I.

One μg of cloning plasmid pSRPDHH DNA (European patent application EP 0909 816 A1) is digested with the restriction enzymes Hind III and EcoRI, and then dephosphorylated with CIP. The resulting dephospsphorylatedpSRPDHH DNA and HindIII-EcoR DNA fragment obtained from pHSG/M1-2-2 areligated using DNA Ligation Kit Version 2.0 (Takara Syuzo, Co. Ltd.).Then, E. coli DH5α is transformed with the ligated DNA and spread ontoLB agar. The transformants obtained are cultured in liquid LB mediumcontaining 100 μg/ml ampicillin, and plasmid DNA is extracted from theresulting culture according to the alkaline-SDS method. The insertionand orientation of the desired DNA fragment in pSRPDHH vector isconfirmed by DNA sequencing using a gene sequence analyzer.

The resulting expression plasmid carrying cDNA encoding the light chainof humanized LM1 TRA-8 is designated pSR/LM1-2.

(4.2) Construction of an Expression Vector for the Light Chain of theHumanized Antibody (LM3 type)

As shown in SEQ ID No. 73 of the Sequence Listing, other humanization(LM3 type) of the amino acid sequences of the light chain of the mouseanti-human DR5 antibody TRA-8 entailed replacing the 8th amino acid(histidine), 9th amino acid (lysine), 10th amino acid (phenylalanine),11th amino acid (methionine), 13th amino acid (threonine), 20th aminoacid (serine), 42nd amino acid (glutamine), 43rd amino acid (serine),77th amino acid (asparagine), 78th amino acid (valine), 80th amino acid(serine) 83rd amino acid (leucine), 85th amino acid (aspartic acid),87th amino acid (phenylalanine), 99th amino acid (glycine) 103rd aminoacid (leucine) and 108th amino acid (alanine) from the N-terminus of theamino acid sequence of the TRA-8 light chain are replaced with proline,serine, serine, leucine, alanine, threonine, lysine, alanine, serine,leucine, proline, phenylalanine, threonine, tyrosine, glutamine, valineand threonine, respectively. The resulting sequence is designated LM3.

Expression plasmids carrying this type of humanized light chain aminoacid sequences of the anti-human DR5 antibody TRA-8 (LM3 type, SEQ IDNo. 73 of the Sequence Listing) are constructed as follows.

1) Synthesis of Primers for Preparing the Variable and Constant Regionsof the Light Chain of Humanized LM3 TRA-8

DNA coding for the LM3 polypeptide chain (SEQ ID No. 73 of the SequenceListing), each of which is a fusion of the variable region of humanizedanti-DR5 antibody TRA-8 light chain and the constant region of the humanIg light chain (κ chain), are respectively synthesized by usingcombinations of PCR.

Further to 7AL1P (SEQ ID No. 47) and 7ALCN (SEQ ID No. 48), thefollowing oligonucleotide primers are synthesized for PCR:

(MOD1F1; SEQ. ID No. 78)5′- atctagttct cagagatgga gacagacaca atcctgctat gggtgctgct gctctgggtt ccagg -3′; (MOD1R1; SEQ ID No. 79)5′- cagcacccat agcaggattg tgtctgtctc catctctgag aactagatga gaggatgctt cttaagctt - 3′; (MOD1F22; SEQ ID No. 80)5′- ctccactggt gacattgtga tgacccaatc tccaagttct ttgtctgcat ctgtggggga cagggtc -3′; (MOD1R22; SEQ ID No. 81)5′- acttggagat tgggtcatca caatgtcacc agtggagcct  ggaacccaga gcag-3′;(MOD1F3; SEQ. ID No. 82) 5′-accatcacct gcaaggccag tcaggatgtg ggtactgctg tagcctggta ccaacagaaa ccaggaa -3′; (MOD1R3; SEQ ID No. 83)5′- tacagcagta cccacatcct gactggcctt gcaggtgatg gtgaccctgt cccccacaga tgcagacaaa ga -3′; (MOD1F42; SEQ ID No. 84)5′- aagcacccaa actcctcatc tattgggcat ccacccggca cactggggtc ccagataggt ttacaggcag t -3′; (MOD1R4; SEQ ID No. 85)5′- cccagtgtgc cgggtggatg cccaatagat gaggagtttg  ggtgcttttc ctggtttctg ttggtaccag gc -3′; (MOD1F5; SEQ ID No. 86)5′- gggtctggga cagacttcac cctcaccatc tctagtctgc agccggagga ttttgcaacc tat -3′; (MOD1R52; SEQ ID No. 87)5′- actagagatg gtgagggtga agtctgtccc agacccactg cctgtaaacc tatctgggac -3′; (MOD1F6; SEQ ID No. 88)5′- tactgtcagc aatatagcag ctatcggacg ttcggtcaag  gcaccaaggt ggaaatc -3′;(MOD1R6; SEQ ID No. 89) 5′- cgtccgatag ctgctatatt gctgacagta ataggttgca aaatcctccg gctgcac -3′ (MOD1F7; SEQ ID No. 90)5′- aaacggactg tggctgcacc atctgtcttc atcttcccgc  catctgatga g -3′;(MOD1R7; SEQ ID No. 91) 5′- gaagatgaag acagatggtg cagccacagt ccgtttgatt tccaccttgg tgccttgacc gaa -3′; and (LR17; SEQ ID No. 101)5′- agatttcaac tgctcatcag atggcgggaa.2) Construction of Plasmid pCR3.1/LM3-3-44 (Cloning of Humanized TRA-8Light Chain Type LM3)

LM3-DNA fragment coding for the amino acid sequence as defined in SEQ IDNo. 73 of the same is prepared by performing 2-step PCR, inserted into aplasmid vector and cloned in E. coli.

a) First Step PCR

LM3-F31B-DNA fragment coding for a secretion signal sequence region witha Hind III restriction enzyme cleavage site added at the 5′-end, FRL₁,CDRL₁, FRL₂, and CDRL₂, FRL₃, CDRL₃, FRL₄ and a portion of the constantregion is prepared under the following conditions.

Composition of the Reaction Solution:

oligonucleotide primer MOD1F1, 5 pmol

oligonucleotide primer MOD1R, 5 pmol

oligonucleotide primer MOD1F22, 5 pmol

oligonucleotide primer MOD1R22, 5 pmol

oligonucleotide primer MOD1F3, 5 pmol

oligonucleotide primer MOD1R3, 5 pmol

oligonucleotide primer MOD1F42, 5 pmol

oligonucleotide primer MOD1R4, 5 pmol

oligonucleotide primer MOD1F5, 5 pmol

oligonucleotide primer MOD1R52, 5 pmol

oligonucleotide primer MOD1F6, 5 pmol

oligonucleotide primer MOD1R6, 5 pmol

oligonucleotide primer MOD1F7, 50 pmol

oligonucleotide primer MOD1R7, 5 pmol

oligonucleotide primer 7AL1P, 50 pmol

oligonucleotide primer LR17, 50 pmol

dNTPs cocktail, 5 μl

10×PCR buffer, 5 μl

ampliTaq DNA polymerase, 2.5 units

The reaction solution having the above composition is adjusted to afinal volume of 50 μl by adding redistilled water and used in PCR.

PCR thermal conditions: Heating at 94° C. for 2 minutes, after which athermal cycle of 94° C. for 1 minute, 55° C. for 1 minute and 72° C. for2 minutes, repeated 30 times, followed by heating at 72° C. for 10minutes.

LM3-F31C-DNA fragment coding for a portion of the constant region withan Eco R I restriction enzyme cleavage site added at the 3′-end isprepared under the following conditions.

The template plasmids, pSRPDHH, is obtained by following the descriptionin a European patent application EP 0 909 816 A1.

Composition of the Reaction Solution:

plasmid pSRPDHH DNA, 25 ng

oligonucleotide primer MOD1F7, 50 pmol

oligonucleotide primer 7ALCN, 50 pmol

dNTPs cocktail, 5 μl

10×PCR buffer, 5 μl

ampliTaq DNA polymerase, 2.5 units

The reaction solution having the above composition is adjusted to afinal volume of 50 μl by adding redistilled water and used in PCR.

PCR thermal conditions: Heating at 94° C. for 2 minutes, after which athermal cycle of 94° C. for 1 minute, 55° C. for 1 minute and 72° C. for2 minutes, repeated 30 times, followed by heating at 72° C. for 10minutes.

The amplified DNA fragments after PCR are separated by 5% polyacrylamidegel electrophoresis. The gel after electrophoresis is stained with 1μg/m of ethidium bromide to detect the produced DNA under UV light. Therespective DNA bands thus detected are excised with a razor.

b) Second Step PCR

LM3-DNA in which above described LM3-F31B-DNA, and LM3-F31C-DNAfragments are fused is prepared under the following conditions.

Composition of the Reaction Solution:

Gel fragment of LM3-F31B-DNA prepared in the first step PCR,

Gel fragment of LM3-F31C-DNA prepared in the first step PCR,

oligonucleotide primer 7AL1P, 50 pmol

oligonucleotide primer 7ALCN, 50 pmol

dNTPs cocktail, 5.0 μl

10×PCR buffer, 5.0 μl

ampliTaq DNA polymerase, 2.5 units

The reaction solution having the above composition is adjusted to afinal volume of 50 μl by adding redistilled water and used in PCR.

PCR thermal conditions: Heating at 94° C. for 2 minutes, after which athermal cycle of 94° C. for 1 minute, 55° C. for 1 minute and 72° C. for2 minutes, repeated 30 times, followed by heating at 72° C. for 10minutes.

The thus prepared LM3-DNA fragment is inserted into plasmid pCR3.1 DNAusing Eukaryotic TA cloning Kit (InVitrogen) following themanufacturer's protocol and introduced into the competent E. ColiTOP10F′ contained in the kit. The nucleotide sequences of these DNAsencoding the light chain of humanized LM3 TRA-8 are confirmed by thedideoxy method (Sanger, F. S., et al., (1977), Proc. Natl. Acad. Sci.USA, 74:5463-5467) using 3700 DNA Analyzer (ABI PRISM®: Perkin ElmerApplied Biosystems. Japan).

The resulting plasmids are designated pCR3.1/LM3-3-44 (the plasmidcarrying cDNA encoding the light chain variable region of humanized LM3TRA-8 and a human Ig light chain constant region).

The obtained plasmid pCR3.1/LM3-3-44 containing LM3-DNA fragment isdigested with the restriction enzymes Hind III and EcoR I.

One μg of cloning plasmid pHSG399 DNA is digested with the restrictionenzymes Hind III and EcoR I, and then dephosphorylated with CIP. Theresulting dephosphorylated pHSG399 DNA and LM3-DNA fragment, that hadbeen digested with the restriction enzymes Hind III and EcoR I, areligated using DNA Ligation Kit Version 2.0 (Takara Syuzo, Co. Ltd.).Then, E. coli DH5α is transformed with the ligated DNA and spread ontoLB agar medium containing 0.1 mM IPTG, 0.1% X-Gal and 50 μg/mlchloramphenicol (final concentrations). The white transformants obtainedare cultured in liquid LB medium containing 50 μg/ml chloramphenicol,and plasmid DNA is extracted from the resulting culture according to thealkaline-SDS method. The extracted plasmid DNA is digested with Hind IIIand EcoR I, and then a clone carrying LM3-DNA fragment is selected by 1%agarose gel electrophoresis.

As a result of the above procedure, plasmid pHSG/M3-3-22 carrying afusion fragment of the variable region of the humanized LM3 TRA-8 lightchain and the constant region of human Igκ chain is obtained. Thetransformant E. coli strain harboring these plasmid, designated as E.coli DH5α/pHSG/M3-3-22 was deposited with International Patent OrganismDepositary, National Institute of Advanced Industrial Science andTechnology, 1-1, Higashi 1 chome Tsukuba-shi, Ibaraki-ken, 305-5466,Japan on Apr. 20, 2001, in accordance with the Budapest Treaty for theDeposit of Microorganisms, and was accorded the accession number FERMBP-7564.

3) Construction of Plasmid pSR/LM3-3-44-10 (Expression Plasmid forHumanized LM3 TRA-8 Light Chain)

The obtained plasmid pHSG/M3-3-22 carrying a fusion fragment of thevariable region of the humanized LM3 TRA-8 light chain and the constantregion of human Igκ chain is digested with the restriction enzymes HindIII and EcoR I.

One μg of cloning plasmid pSRPDHH DNA (European patent application EP 0909 816 A1) is digested with the restriction enzymes Hind III and EcoRI, and then dephosphorylated with CIP. The resulting dephosphorylatedpSRPDHH DNA and HindIII-EcoRI DNA fragment obtained from pHSG/M3-3-22are ligated using DNA Ligation Kit Version 2.0 (Takara Syuzo. Co. Ltd.).Then, E. coli DH5α is transformed with the ligated DNA and spread ontoLB agar. The transformants obtained are cultured in liquid LB mediumcontaining 100 μg/ml ampicillin, and plasmid DNA is extracted from theresulting culture according to the alkaline-SDS method. The insertionand orientation of the desired DNA fragment in pSRPDHH vector isconfirmed by DNA sequencing using a gene sequence analyzer (ABI PRIDM®3700 DNA Analyzer; Applied Biosystems).

The resulting expression plasmid carrying cDNA encoding the light chainof humanized LM3 TRA-8 is designated pSR/LM3-3-44-10.

(4.3) Construction of an Expression Vector for the Light Chain of theHumanized Antibody (LM4 Type)

As shown in SEQ ID No. 74 of the Sequence Listing, other humanization(LM4 type) of the amino acid sequences of the light chain of the mouseanti-human DR5 antibody TRA-8 entailed replacing the 8th amino acid(histidine), 9th amino acid (lysine), 10th amino acid (phenylalanine),11th amino acid (methionine), 13th amino acid (threonine), 20th aminoacid (serine), 42nd amino acid (glutamine), 43rd amino acid (serine),77th amino acid (asparagine), 78th amino acid (valine), 80th amino acid(serine) 83rd amino acid (leucine), 85th amino acid (aspartic acid),99th amino acid (glycine) 103rd amino acid (leucine) and 108th aminoacid (alanine) from the N-terminus of the amino acid sequence of theTRA-8 light chain are replaced with proline, serine, serine, leucine,alanine, threonine, lysine, alanine, serine, leucine, proline,phenylalanine, threonine, glutamine, valine and threonine respectively.The resulting sequence is designated LM4. Expression plasmids carryingthis type of humanized light chain amino acid sequences of theanti-human DR5 antibody TRA-8 (LM4 type) (SEQ ID No. 74 of the SequenceListing) are constructed as follows.

1) Synthesis of Primers for Preparing the Variable and Constant Regionsof the Light Chain of Humanized LM4 TRA-8

DNA coding for the LM4 polypeptide chain (SEQ ID No. 74 of the SequenceListing), each of which is a fusion of the variable region of humanizedanti-DR5 antibody TRA-8 light chain and the constant region of the humanIg light chain (K chain), are respectively synthesized by usingcombinations of PCR.

Further to 7AL1P (SEQ ID No. 47), 7ALCN (SEQ ID No. 48), MOD1F1 (SEQ IDNo. 78), MOD1R1 (SEQ ID No. 79), MOD1F22 (SEQ ID No. 80), MOD1R22 (SEQID No. 81), MOD1F3 (SEQ ID No. 82), MOD1R3 (SEQ ID No. 83), MOD1F42 (SEQID No. 84), MOD1R4 (SEQ ID No. 85), MOD1F5 (SEQ ID No. 86), MOD1R52 (SEQID No. 87), MOD1F7 (SEQ ID No. 90), and MOD1R7 (SEQ ID No. 91), LR17(SEQ ID No. 101), the following oligonucleotide primers are synthesizedfor PCR:

(MOD1F62; SEQ ID No. 92) 5′- ttctgtcagc aatatagcag ctatcggacg ttcggtcaaggcaccaaggt ggaaatc -3′ (MOD1R62; SEQ ID No. 93)5′- cgtccgatag ctgctatatt gctgacagaa ataggttgca aaatcctccg gctgcag -3′2) Construction of Plasmid pCR3.1/LM4-5-3 (Cloning of Humanized TRA-8Light Chain Type LM4)

LM4-DNA fragment coding for the amino acid sequence as defined in SEQ IDNo. 74 of the same is prepared by performing 2-step PCR, inserted into aplasmid vector and cloned in E. coli.

a) First Step PCR

LM4-F41B-DNA fragment coding for a secretion signal sequence region witha Hind III restriction enzyme cleavage site added at the 5′-end, FRL₁,CDRL₁, FRL₂, and CDRL₂, FRL₃, CDRL₃, FRL₄ and a portion of the constantregion is prepared under the following conditions.

Composition of the Reaction Solution:

oligonucleotide primer MOD1F1, 5 pmol

oligonucleotide primer MOD1R1, 5 pmol

oligonucleotide primer MOD1F22, 5 pmol

oligonucleotide primer MOD1R22, 5 pmol

oligonucleotide primer MOD1F3, 5 pmol

oligonucleotide primer MOD1R3, 5 pmol

oligonucleotide primer MOD1F42, 5 pmol

oligonucleotide primer MOD1R4, 5 pmol

oligonucleotide primer MOD1F5, 5 pmol

oligonucleotide primer MOD1R52, 5 pmol

oligonucleotide primer MOD1F62, 5 pmol

oligonucleotide primer MOD1R62, 5 pmol

oligonucleotide primer MOD1F7, 50 pmol

oligonucleotide primer MOD1R7, 5 pmol

oligonucleotide primer 7AL1P, 50 pmol

oligonucleotide primer LR17, 50 pmol

dNTPs cocktail, 5 μl

10×PCR buffer, 5 μl

ampliTaq DNA polymerase, 2.5 units

The reaction solution having the above composition is adjusted to afinal volume of 50 μl by adding redistilled water and used in PCR.

PCR thermal conditions: Heating at 94° C. for 2 minutes, after which athermal cycle of 94° C. for 1 minute, 55° C. for 1 minute and 72° C. for2 minutes, repeated 30 times, followed by heating at 72° C. for 10minutes.

LM4-F41C-DNA fragment coding for a portion of the constant region withan Eco R I restriction enzyme cleavage site added at the 3′-end isprepared under the following conditions.

The template plasmids, pSRPDHH, are obtained by following thedescription in a European patent application EP 0 909 816 A1.

Composition of the Reaction Solution:

plasmid pSRPDHH DNA, 25 ng

oligonucleotide primer MOD1F7, 50 pmol

oligonucleotide primer 7ALCN, 50 pmol

dNTPs cocktail, 5 μl

10×PCR buffer, 5 μl

ampliTaq DNA polymerase, 2.5 units

The reaction solution having the above composition is adjusted to afinal volume of 50 μl by adding redistilled water and used in PCR.

PCR thermal conditions: Heating at 94° C. for 2 minutes, after which athermal cycle of 94° C. for 1 minute, 55° C. for 1 minute and 72° C. for2 minutes, repeated 30 times, followed by heating at 72° C. for 10minutes.

The amplified DNA fragments after PCR are separated by 5% polyacrylamidegel electrophoresis. The gel after electrophoresis is stained with 1μg/ml of ethidium bromide to detect the produced DNA under UV light. Therespective DNA bands thus detected are excised with a razor blade.

b) Second Step PCR

LM4-DNA in which above described LM4-F41B-DNA, and LM4-F41C-DNAfragments are fused is prepared under the following conditions.

Composition of the Reaction Solution:

Gel fragment of LM4-F41B-DNA prepared in the first step PCR,

Gel fragment of LM4-F41C-DNA prepared in the first step PCR,

oligonucleotide primer 7AL1P, 50 pmol

oligonucleotide primer 7ALCN, 50 pmol

dNTPs cocktail, 5.0 μl

10×PCR buffer, 5.0 μl

ampliTaq DNA polymerize, 2.5 units

The reaction solution having the above composition is adjusted to afinal volume of 50 μl by adding redistilled water and used in PCR.

PCR thermal conditions: Heating at 94° C. for 2 minutes, after which athermal cycle of 94° C. for 1 minute, 55° C. for 1 minute and 72° C. for2 minutes, repeated 30 times, followed by heating at 72° C. for 10minutes.

The thus prepared LM4-DNA fragment is inserted into plasmid pCR3.1DNAusing Eukaryotic TA cloning Kit (InVitrogen) following themanufacturer's protocol and introduced into the competent E. ColiTOP10F′ contained in the kit. The nucleotide sequences of these DNAsencoding the light chain of humanized LM4 TRA-8 are confirmed by thedideoxy method (Sanger, F. S., et al., (1977), Proc. Natl. Acad. Sci.USA, 74:5463-5467) using 3700 DNA Analyzer (ABI PRISM®; Perkin ElmerApplied Biosystems, Japan).

The resulting plasmids are designated pCR3.1/LM4-5-3 (the plasmidcarrying cDNA encoding the light chain variable region of humanized LM4TRA-8 and a human Ig light chain constant region).

The obtained plasmid pCR3.1/LM4-5-3 containing LM4-DNA fragment isdigested with the restriction enzymes Hind III and EcoR I.

One μg of cloning plasmid pHSG399 DNA is digested with the restrictionenzymes Hind III and EcoR I, and then dephosphorylated with CIP. Theresulting dephosphorylated pHSG399 DNA and LM4-DNA fragment, that hadbeen digested with the restriction enzymes Hind III and EcoR I, areligated using DNA Ligation Kit Version 2.0 (Takara Syuzo, Co. Ltd.).Then, E. coli DH5α is transformed with the ligated DNA and spread ontoLB agar medium containing 0.1 mM IPTG, 0.1% X-Gal and 50 μg/mlchloramphenicol (final concentrations). The white transformants obtainedare cultured in liquid LB medium containing 50 μg/ml chloramphenicol,and plasmid DNA is extracted from the resulting culture according to thealkaline-SDS method. The extracted plasmid DNA is digested with Hind IIIand EcoR I, and then a clone carrying LM4-DNA fragment is selected by 1%agarose gel electrophoresis.

As a result of the above procedure, plasmid pHSG/M4-5-3-1 carrying afusion fragment of the variable region of the humanized LM4 TRA-8 lightchain and the constant region of human Igκ chain is obtained. Thetransformant E. coli strain harboring these plasmid, designated as E.coli DH5α/pHSG/M4-5-3-1 was deposited with International Patent OrganismDepositary, National Institute of Advanced Industrial Science andTechnology, 1-1, Higashi 1 chome Tsukuba-shi, Ibaraki-ken, 305-5466,Japan on Apr. 20, 2001, in accordance with the Budapest Treaty for theDeposit of Microorganisms and was accorded the accession number FERMBP-7565.

3) Construction of Plasmid pSR/LM4-5-3-3 (Expression Plasmid forHumanized LM4 TRA-8 Light Chain)

The obtained plasmid pHSG/M4-5-3-1 carrying a fusion fragment of thevariable region of the humanized LM4 TRA-8 light chain and the constantregion of human Igκ chain is digested with the restriction enzymes HindIII and EcoR I.

One μg of cloning plasmid pSRPDHH DNA (European patent application EP 0909 816 A1) is digested with the restriction enzymes Hind III and EcoRI, and then dephosphorylated with CIP. The resulting dephosphorylatedpSRPDHH DNA and HindIII-EcoRI DNA fragment obtained from pHSG/M4-5-3-1are ligated using DNA Ligation Kit Version 2.0 (Takara Syuzo, Co. Ltd.).Then, E. coli DH5α is transformed with the ligated DNA and spread ontoLB agar. The transformants obtained are cultured in liquid LB mediumcontaining 100 μg/ml ampicillin, and plasmid DNA is extracted from theresulting culture according to the alkaline-SDS method. The insertionand orientation of the desired DNA fragment in pSRPDHH vector isconfirmed by DNA sequencing using a gene sequence analyzer (ABI PRISM®3700 DNA Analyzer; Applied Biosystems).

The resulting expression plasmid carrying cDNA encoding the light chainof humanized LM4 TRA-8 is designated pSR/LM4-5-3-3.

(4.4) Construction of an Expression Vector for the Light Chain of theHumanized Antibody (LM5 Type)

As shown in SEQ ID No. 75 of the Sequence Listing, other humanization(LM5 type) of the amino acid sequences of the light chain of the mouseanti-human DR5 antibody TRA-8 entailed replacing the 8th amino acid(histidine), 9th amino acid (lysine), 10th amino acid (phenylalanine),11th amino acid (methionine), 13th amino acid (threonine), 20th aminoacid (serine), 42nd amino acid (glutamine), 43rd amino acid (serine),77th amino acid (asparagine), 78th amino acid (valine), 80th amino acid(serine) 83rd amino acid (leucine), 103rd amino acid (leucine) and 108thamino acid (alanine) from the N-terminus of the amino acid sequence ofthe TRA-8 light chain are replaced with proline, serine, serine,leucine, alanine, threonine, lysine, alanine, serine, leucine, proline,phenylalanine, valine and threonine respectively. The resulting sequenceis designated LM5.

Expression plasmids carrying this type of humanized light chain aminoacid sequences of the anti-human DR5 antibody TRA-8 (LM5 type) (SEQ IDNo. 75 of the Sequence Listing) is constructed as follows.

1) Synthesis of Primers for Preparing the Variable and Constant Regionsof the Light Chain of Humanized LM5 TRA-8

DNA coding for the LM5 polypeptide chain (SEQ ID No. 75 of the SequenceListing), each of which is a fusion of the variable region of humanizedanti-DR5 antibody TRA-8 light chain and the constant region of the humanIg light chain (κ chain), are respectively synthesized by usingcombinations of PCR.

Further to 7AL1P (SEQ ID No. 47), 7ALCN (SEQ ID No. 48), MOD1F1 (SEQ IDNo. 78), MOD1R1 (SEQ ID No. 79), MOD1F22 (SEQ ID No. 80), MOD1R22 (SEQID No. 81), MOD1F3 (SEQ ID No. 82), MOD1R3 (SEQ ID No. 83), MOD1F42 (SEQID No. 84), MOD1R4 (SEQ ID No. 85), MOD1R52 (SEQ ID No. 87), MOD1F7 (SEQID No. 90), and LR17 (SEQ ID No. 101), the following oligonucleotideprimers are synthesized for PCR:

(MOD1F52; SEQ ID No. 94) 5′- gggtctggga cagacttcac cctcaccatc tctagtctgcagccggagga ttttgcagat tat-3′ (MOD1F63; SEQ ID No. 95)5′- ttctgtcagc aatatagcag ctatcggacg ttcggtggag gcaccaaggt ggaaatc -3′(MOD1R63; SEQ ID No. 96) 5′- cgtccgatag ctgctatatt gctgacagaa ataatctgcaaaatcctccg gctgcag -3′ (MOD1R72; SEQ ID No. 102)5′- gaagatgaag acagatggtg cagccacagt ccgtttgatttccaccttgg tgcctccacc gaa-3′2) Construction of Plasmid pCR3.1/LMS-3-42 (Cloning of Humanized TRA-8Light Chain Type LM5)

LM5-DNA fragment coding for the amino acid sequence as defined in SEQ IDNo. 75 of the same is prepared by performing 2-step PCR, inserted into aplasmid vector and cloned in E. coli.

a) First Step PCR

LM5-F51B-DNA fragment coding for a secretion signal sequence region witha Hind III restriction enzyme cleavage site added at the 5′-end, FRL₁,CDRL₁, FRL₂, CDRL₂, FRL₃, CDRL₃, FRL₄ and a portion of the constantregion is prepared under the following conditions.

Composition of the Reaction Solution:

oligonucleotide primer MOD1F1, 5 pmol

oligonucleotide primer MOD1R1, 5 pmol

oligonucleotide primer MOD1F22, 5 pmol

oligonucleotide primer MOD1R22, 5 pmol

oligonucleotide primer MOD1F3, 5 pmol

oligonucleotide primer MOD1R3, 5 pmol

oligonucleotide primer MOD1F42, 5 pmol

oligonucleotide primer MOD1R4, 5 pmol

oligonucleotide primer MOD1F52, 5 pmol

oligonucleotide primer MOD1R52, 5 pmol

oligonucleotide primer MOD1F63, 5 pmol

oligonucleotide primer MOD1R63, 5 pmol

oligonucleotide primer MOD1F7, 50 pmol

oligonucleotide primer MOD1R72, 5 pmol

oligonucleotide primer 7AL1P, 50 pmol

oligonucleotide primer LR17, 50 pmol

dNTPs cocktail, 5 μl

10×PCR buffer, 5 μl

ampliTaq DNA polymerase, 2.5 units

The reaction solution having the above composition, is adjusted to afinal volume of 50 μl by adding redistilled water and used in PCR.

PCR thermal conditions: Heating at 94° C. for 2 minutes, after which athermal cycle of 94° C. for 1 minute, 55° C. for 1 minute and 72° C. for2 minutes, repeated 30 times, followed by heating at 72° C. for 10minutes.

LM5-F51C-DNA fragment coding for a portion of the constant region withan Eco R I restriction enzyme cleavage site added at the 3′-end isprepared under the following conditions. The template plasmids, pSRPDHH,is obtained by following the description in an European patentapplication EP 0 909 816 A1.

Composition of the Reaction Solution:

plasmid pSRPDHH DNA, 25 ng

oligonucleotide primer MOD1F7, 50 pmol

oligonucleotide primer 7ALCN, 50 pmol

dNTPs cocktail, 5 μl

10×PCR buffer, 5 μl

ampliTaq DNA polymerase, 2.5 units

The reaction solution having the above composition is adjusted to afinal volume of 50 μl by adding redistilled water and used in PCR.

PCR thermal conditions: Heating at 94° C. for 2 minutes, after which athermal cycle of 94° C. for 1 minute, 55° C. for 1 minute and 72° C. for2 minutes, repeated 30 times, followed by heating at 72° C. for 10minutes.

The amplified DNA fragments after PCR are separated by 5% polyacrylamidegel electrophoresis. The gel after electrophoresis is stained with 1μg/ml of ethidium bromide to detect the produced DNA under UV light. Therespective DNA bands thus detected are excised with a razor blade.

b) Second Step PCR

LM5-DNA in which above described LM5-F51B-DNA, and LM5-F51C-DNAfragments are fused is prepared under the following conditions.

Composition of the Reaction Solution:

Gel fragment of LM5-F51B-DNA prepared in the first step PCR,

Gel fragment of LM5-F51C-DNA prepared in the first step PCR,

oligonucleotide primer 7AL1P, 50 pmol

oligonucleotide primer 7ALCN, 50 pmol

dNTPs cocktail, 5.0 μl

10×PCR buffer, 5.0 μl

ampliTaq DNA polymerase, 2.5 units

The reaction solution having the above composition is adjusted to afinal volume of 50 μl by adding redistilled water and used in PCR.

PCR thermal conditions: Heating at 94° C. for 2 minutes, after which athermal cycle of 94° C. for 1 minute, 55° C. for 1 minute and 72° C. for2 minutes, repeated 30 times, followed by heating at 72° C. for 10minutes.

The thus prepared LM5-DNA fragment is inserted into plasmid pCR3.1DNAusing Eukaryotic TA cloning Kit (InVitrogen) following themanufacturer's protocol and introduced into the competent E. ColiTOP10F′ contained in the kit. The nucleotide sequences of these DNAsencoding the light chain of humanized LM5 TRA-8 are confirmed by thedideoxy method (Sanger. F. S., et al., (1977), Proc. Natl. Acad. Sci.USA, 74:5463-5467) using DNA analyzer.

The resulting plasmids are designated pCR3.1/LM5-3-42 (the plasmidcarrying cDNA encoding the light chain variable region of humanized LM5TRA-8 and a human Ig light chain constant region).

The obtained plasmid pCR3.1/LM5-3-42 containing LM5-DNA fragment isdigested with the restriction enzymes Hind III and EcoR I.

One μg of cloning plasmid pHSG399 DNA is digested with the restrictionenzymes Hind III and EcoR I, and then dephosphorylated with CIP. Theresulting dephosphorylated pHSG399 DNA and LM5-DNA fragment, that hadbeen digested with the restriction enzymes Hind III and EcoR I, areligated using DNA Ligation Kit Version 2.0 (Takara Syuzo, Co. Ltd.).Then, E. coli DH5α is transformed with the ligated DNA and spread ontoLB agar medium containing 0.1 mM IPTG, 0.1% X-Gal and 50 μg/mlchloramphenicol (final concentrations). The white transformants obtainedare cultured in liquid LB medium containing 50 μg/ml chloramphenicol,and plasmid DNA is extracted from the resulting culture according to thealkaline-SDS method. The extracted plasmid DNA is digested with Hind IIIand EcoR I, and then a clone carrying LM5-DNA fragment is selected by 1%agarose gel electrophoresis.

As a result of the above procedure, plasmid pHSG/M5-3-27 carrying afusion fragment of the variable region of the humanized LM5 TRA-8 lightchain and the constant region of human Igκ chain is obtained.

3) Construction of Plasmid pSR/LM5-3-27-1 (Expression Plasmid forHumanized LM5 TRA-8 Light Chain)

The obtained plasmid pHSG/M5-3-27 carrying a fusion fragment of thevariable region of the humanized LM5 TRA-8 light chain and the constantregion of human Igκ chain is digested with the restriction enzymes HindIII and EcoR I.

One μg of cloning plasmid pSRPDHH DNA (European patent application EP 0909 816 A1) is digested with the restriction enzymes Hind III and EcoRI, and then dephosphorylated with CIP. The resulting dephosphorylatedpSRPDHH DNA and HindIII-EcoRI DNA fragment obtained from pHSG/M5-3-27are ligated using DNA Ligation Kit Version 2.0 (Takara Syuzo, Co. Ltd.).Then, E. coli DH5α is transformed with the ligated DNA and spread ontoLB agar. The transformants obtained are cultured in liquid LB mediumcontaining 100 μg/ml ampicillin, and plasmid DNA is extracted from theresulting culture according to the alkaline-SDS method. The insertionand orientation of the desired DNA fragment in pSRPDHH vector isconfirmed by DNA sequencing using a gene sequence analyzer (ABI PRISM®3700 DNA Analyzer: Applied Biosystems).

The resulting expression plasmid carrying cDNA encoding the light chainof humanized LM5 TRA-8 is designated pSR/LM5-3-27-1.

(4.5) Construction of an Expression Vector for the Light Chain of theHumanized Antibody (Chimera Type)

The sequence shown in SEQ ID No. 76 of the Sequence Listing, the aminoacid sequence of the light chain of chimera type TRA-8, is designatedLM6.

Expression plasmids carrying this type of humanized light chain aminoacid sequences of the anti-human DR5 antibody TRA-8 (LM6 type) (SEQ IDNo. 75 of the Sequence Listing) is constructed as follows.

1) Synthesis of Primers for Preparing the Variable and Constant Regionsof the Light Chain of Humanized LM6 TRA-8

DNA coding for the LM6 polypeptide chain (SEQ ID No. 75 of the SequenceListing), each of which is a fusion of the variable region of mouseanti-DR5 antibody TRA-8 light chain (LM6 type) and the constant regionof the human Ig light chain (κ chain), are respectively synthesized byusing combinations of PCR.

Further to 7AL1P (SEQ ID No. 47) and 7ALCN (SEQ ID No. 48), thefollowing oligonucleotide primers are synthesized for PCR:

(HKSPR13; SEQ ID No. 97) 5′- tgatgtggac atgaatttgt gagactgggt catcacaatgtcaccagtgg a -3′; (MVF11; SEQ ID No. 98)5′- tgggttccag gctccactgg tgacattgtg atgacccagt ctcacaaatt c -3′;(MVR11; SEQ ID No. 99) 5′- aagacagatg gtgcagccac agcccgtttg atttccagcttggtgcctc -3′; and (MCF11; SEQ ID No. 100)5′- aagctggaaa tcaaacgggc tgtggctgca ccatctgtct tcatc -3′.2) Construction of Plasmid pCR3.1/LM6-1-16 (Cloning of Humanized TRA-8Light Chain Type LM6)

LM6-DNA fragment coding for the amino acid sequence as defined in SEQ IDNo. 75 of the same is prepared by performing 2-step PCR, inserted into aplasmid vector and cloned in E. coli.

a) First Step PCR

LM6-F1-DNA fragment coding for a secretion signal sequence and a portionof FRL₁ region with a Hind III restriction enzyme cleavage site added atthe 5′-end is prepared under the following conditions. The templateplasmids, pHSGHM17 and pSRPDHH, are obtained by following thedescription in a European patent application EP 0 909 816 A1.

Composition of the Reaction Solution:

plasmid pHSGHM17 DNA, 25 ng

oligonucleotide primer 7AL1P, 50 pmol

oligonucleotide primer HKSPR13, 50 pmol

dNTPs cocktail, 5 μl

10×PCR buffer, 5 μl

ampliTaq DNA polymerase (PerkinElmer), 2.5 units

The reaction solution having the above composition is adjusted to afinal volume of 50 μl by adding redistilled water and used in PCR.

PCR thermal conditions: Heating at 94° C. for 2 minutes, after which athermal cycle of 94° C. for 1 minute, 55° C. for 1 minute and 72° C. for2 minutes, repeated 30 times, followed by heating at 72° C. for 10minutes.

LM6-F2-DNA fragment coding for a portion of FRL₁, CDRL₁, FRL₂, CDRL₂,FRL₃, CDRL₃, FRL₄ and a portion of the constant region is prepared underthe following conditions.

Composition of the Reaction Solution:

plasmid pL28 DNA, 25 ng

oligonucleotide primer MVF11, 50 pmol

oligonucleotide primer MVR12, 50 pmol

dNTPs cocktail, 5 μl

10×PCR buffer, 5 μl

ampliTaq DNA polymerase, 2.5 units

The reaction solution having the above composition is adjusted to afinal volume of 50 μl by adding redistilled water and used in PCR.

PCR thermal conditions: Heating at 94° C. for 2 minutes, after which athermal cycle of 94° C. for 1 minute, 55° C. for 1 minute and 72° C. for2 minutes, repeated 30 times, followed by heating at 72° C. for 10minutes.

LM6-F3-DNA fragment coding for a portion of FRL₄ and the constant regionwith an EcoR I restriction enzyme cleavage site added at the 3′-end isprepared under the following conditions.

Composition of the Reaction Solution:

plasmid pSRPDHH DNA, 25 ng

oligonucleotide primer MCF11, 50 pmol

oligonucleotide primer 7ALCN, 50 pmol

dNTPs cocktail, 5 μl

10×PCR buffer, 5 μl

ampliTaq DNA polymerase, 2.5 units

The reaction solution having the above composition is adjusted to afinal volume of 50 μl by adding redistilled water and used in PCR.

PCR thermal conditions: Heating at 94° C. for 2 minutes, after which athermal cycle of 94° C. for 1 minute, 55° C. for 1 minute and 72° C. for2 minutes, repeated 30 times, followed by heating at 72° C. for 10minutes.

The amplified DNA fragments after PCR are separated by 5% polyacrylamidegel electrophoresis. The gel after electrophoresis is stained with 1μg/ml of ethidium bromide to detect the produced DNA under UV light. Therespective DNA bands thus detected are excised with a razor blade.

b) Second Step PCR

LM6-DNA in which above described LM6-F1-DNA, LM6-F2-DNA, and LM6-F3-DNAfragments are fused is prepared under the following conditions.

Composition of the Reaction Solution:

Gel fragment of LM6-F1-DNA prepared in the first step PCR,

Gel fragment of LM6-F2-DNA prepared in the first step PCR,

Gel fragment of LM6-F3-DNA prepared in the first step PCR,

oligonucleotide primer 7AL1P, 50 pmol

oligonucleotide primer 7ALCN, 50 pmol

dNTPs cocktail, 5.0 μl

10×PCR buffer, 5.0 μl

ampliTaq DNA polymerase, 2.5 units

The reaction solution having the above composition is adjusted to afinal volume of 50 μl by adding redistilled water and used in PCR.

PCR thermal conditions: Heating at 94° C. for 2 minutes, after which athermal cycle of 94° C. for 1 minute, 55° C. for 1 minute and 72° C. for2 minutes, repeated 30 times, followed by heating at 72° C. for 10minutes.

The thus prepared LM6-DNA fragment is inserted into plasmid pCR3.1DNAusing Eukaryotic TA cloning Kit (Invitrogen) following themanufacturer's protocol and introduced into the competent E. ColiTOP10F′ contained in the kit. The nucleotide sequences of these DNAsencoding the light chain of humanized TRA-8 are confirmed by the dideoxymethod using a DNA analyzer.

The resulting plasmids are designated pCR3.1/LM6-1-16 (the plasmidcarrying cDNA encoding the light chain variable region of mouse TRA-8and a human Ig light chain constant region).

The obtained plasmid pCR3.1/LM6-1-16 containing LM6-DNA fragment isdigested with the restriction enzymes Hind III and EcoR I.

One μg of cloning plasmid pHSG399 DNA is digested with the restrictionenzymes Hind III and EcoR I, and then dephosphorylated with CIP. Theresulting dephosphorylated pHSG399 DNA and LM6-DNA fragment, that hadbeen digested with the restriction enzymes Hind III and EcoR I, areligated using DNA ligation Kit Version 2.0 (Takara Syuzo, Co. Ltd.).Then, E. coli DH5α is transformed with the ligated DNA and spread ontoLB agar medium containing 0.1 mM IPTG, 0.1% X-Gal and 50 μg/mlchloramphenicol (final concentrations). The white transformants obtainedare cultured in liquid LB medium containing 50 μg/ml chloramphenicol,and plasmid DNA is extracted from the resulting culture according to thealkaline-SDS method. The extracted plasmid DNA is digested with Hind IIIand EcoR I, and then a clone carrying LM6-DNA fragment is selected by 1%agarose gel electrophoresis.

As a result of the above procedure, plasmid pHSG/M6-1-4-1 carrying afusion fragment of the variable region of the mouse TRA-8 light chainand the constant region of human Igκ chain is obtained. The transformantE. coli strain harboring these plasmid, designated as E. coliDH5α/pHSG/M6-1-4-1 was deposited with International Patent OrganismDepositary, National Institute of Advanced Industrial Science andTechnology, 1-1, Higashi 1 chome Tsukuba-shi, Ibaraki-ken, 305-5466,Japan on Apr. 20, 2001, in accordance with the Budapest Treaty for theDeposit of Microorganisms, and was accorded the accession number FERMBP-7566.

3) Construction of Plasmid pSR/LM6-1-4-6 (Expression Plasmid for ChimeraType LM6 TRA-8 Light Chain)

The obtained plasmid pHSG/LM6-1-4-1 carrying a fusion fragment of thevariable region of the mouse TRA-8 light chain and the constant regionof human Igκ chain is digested with the restriction enzymes Hind III andEcoR I.

One μg of cloning plasmid pSRPDHH DNA is digested with the restrictionenzymes Hind III and EcoR I, and then dephosphorylated with CIP. Theresulting dephosphorylated pSRPDHH DNA and HindIII-EcoRI DNA fragmentobtained from pHSG/LM6-1-4-1 are ligated using DNA Ligation Kit Version2.0 (Takara Syuzo, Co. Ltd.). Then, E. coli DH5α is transformed with theligated DNA and spread onto LB agar. The transformants obtained arecultured in liquid LB medium containing 100 μg/ml ampicillin, andplasmid DNA is extracted from the resulting culture according to thealkaline-SDS method. The insertion and orientation of the desired DNAfragment in the vector is confirmed by DNA sequencing using a genesequence analyzer.

The resulting expression plasmid carrying cDNA encoding the light chainof TRA-8 (chimera type) is designated pSR/LM6-1-4-6.

(5) Production of Several Types-Humanized or Chimeric TRA-8 Antibody

Transfection of COS-7 cells is conducted by FUGENE6 transfection reagentmethods (Boehringer Mannheim Biochemica) according to the instructionmanual provided with the kit.

COS-7 cells (American Type Culture Collection No. CRL-1651) are grown tosemi-confluent (3×10⁶ cells/dish) in a culture dish (culture area: 57cm²; Sumitomo Bakelite) containing Dulbecco's Modified Eagle medium(hereinafter referred to as “D-MED”; Gibco BRL) supplemented with 10%fetal calf serum (hereinafter abbreviated as “FCS”; Moregate).

In the meantime, 10 μg/dish (total 5 dishes) of the humanized DR5 heavychain expression plasmid DNA (pHA15-1) and 10 μg/dish of the humanizedDR5 light chain expression plasmid DNA prepared by the alkaline-SDSmethod and cesium chloride density gradient centrifugation are mixed,and then precipitated with ethanol, followed by suspending in 5 μl/dishof dH₂O.

After 15 μl/dish of FUGENE6 Transfection regent is mixed with 180μl/dish D-MEM without FCS, this FUGENE solution (185 μl/dish) is mixedwith 5 μl/dish DNA solution containing 10 μg/dish of the humanized DR5heavy chain expression plasmid DNA and 10 μg/dish of the humanized DR5light chain expression plasmid DNA. After 15 minutes incubation at roomtemperature, the obtained plasmid suspension (200 μl) is added to thepreviously prepared COS-7 plates. After incubating in 5% CO₂ at 37° C.for 24 hours, the culture medium is changed with D-MEM without FCS.After incubating in 5% CO₂ at 37° C. for 72 hours, the culturesupernatant is recovered to purify the expression products in thesupernatant fluids. By the method as described above, COS-7 cells aretransfected with each of the following plasmid combinations:

(A): cotransfection of pHA15-1 and pSR/LM1-2 (H1L1)

(B): cotransfection of pHB14-1 and pSR/M2-1 (H2L2)

(C): cotransfection of pHB14-1 and pSR/LM3-3-44-10 (H2L3)

(D): cotransfection of pHB14-1 and pSR/LM4-5-3-3 (H2L4)

(E): cotransfection of pHC10-3 and pSR/M2-1 (H3L2)

(F): cotransfection of pHC10-3 and pSR/LM3-3-44-10 (H3L3)

(G): cotransfection of pHC110-3 and pSR/LM4-5-3-3 (H3L4)

(H): cotransfection of pHD21-1 and pSR/LM5-3-27-1 (H4L5)

(I): cotransfection of pM11-1 and pSR/LM6-1-4-6 (Chimera)

The culture is then centrifuged (3,500 r.p.m., 15 minutes) and collectedthe supernatant. The supernatant is filtrated with 0.45 μm filter(ADVANTEC TOYO DISMIC-25cs, Cat #25CS045 AS). The purification of IgGfrom the filtrates are achieved using Protein G-POROS affinitychromatography (Applied Biosystems) under the following conditions:

HPLC system: BioCAD 700E (Applied Biosystems)

column: ProteinG-ID sensor cartridge

(column size: 2.1 mmID×30 mm LD, bed volume: 0.1 ml; Applied Biosystems)

elution buffer: 0.1 M Glycine-HCl (pH 2.5)

neutralization buffer: 1 M Tris-HCl (pH 8.5)

detection: 280 nm

flow rate: 1 ml/min

fraction size: 0.5 ml/0.5 min

fraction tube: 1.5 ml polypropylene microtube

temperature: 4° C.

After all the filtrates are applied to column, 50 ml of PBS (Sigma, Cat#1000-3) is used to wash column. When the elution buffer is applied,fraction collector started. Each fraction microtube previously contained55 μl of 1 M NaCl, 110 μl of neutralization buffer and 74 μl of 2 mg/mlbovine serum albumin (Sigma, Cat #A-7030) in PBS. The fractions from No.7 through No. 8 are collected.

Verification of the expression of the humanized antibodies andquantitative assay of the expression products in the culture supernatantfluids prepared is performed by ELISA with an antibody againstanti-human IgG.

To each well of a 96-well plate (MaxiSorp, Nunc), 100 μl of goatanti-human IgG Fc specific polyclonal antibody (Kappel) dissolved at thefinal concentration of 0.5 μg/ml in adsorption buffer (0.05 M sodiumhydrogencarbonate, 0.02% sodium azide, pH 9.6) is added and the plate isincubated at 37° C. for 2 hours to cause adsorption of the antibody.Then, the plate is washed with 350 μl of PBS-T five times. To the wellsafter washing, the culture supernatant diluted with D-MEM containing 10%FCS is added and incubated at 37° C. for 2 hours. After washing againwith PBS-T, 100 μl of alkaline phosphatase-labeled goat anti-human IgGFc specific polyclonal antibody (Jackson immuno Research Lab.) diluted10,000-fold with PBS-T is added to each well and incubated at 37° C. for2 hours. After washing again with PBS-T, a substrate solution ofp-nitrophenyl phosphate obtained from Alkaline Phosphatase Substrate kit(Bio Rad) is added according to the instruction manual provided with thekit. After incubating at 37° C. for 0.5 to 1 hour, the absorbance at 405nm is measured. In the present experiments, human plasma immunoglobulinG subclass 1 (IgG1) (Biopure AG) diluted with D-MEM containing 10% FCSto certain concentrations is used as concentration reference samples ofthe humanized DR5 antibodies contained in the culture supernatantfluids.

As a result, the expression and purified products in the culturesupernatant are detected specifically with the anti-human IgG antibody.The final concentration of human IgG antibody is 44.03 μg/ml (H1L1),39.8 μg/ml (H2L2), 26.7 μg/ml (H2L3), 41.0 μg/ml (H2L4), 39.3 μg/ml(H3L2), 24.7 μg/ml (H3L3), 21.5 μg/ml (H3L4), 16.7 μg/ml (H4L5) and 18.3μg/ml (chimera), respectively.

(6) Apoptosis-Inducing Activity of Several Types Humanized Antibody orChimeric Antibody

Jurkat cells (ATCC No. TIB-152), are used to examine theapoptosis-inducing activity of the purified humanized TRA-8 antibody.

Jurkat cells cultured in RPMI1640 medium with 10% FCS (Gibco BRL) at 37°C. for 3 days in the presence of 5% CO₇, are dispensed into each well ofa 96-well microplate (Sumitomo Bakelite) at 50 μl per well. Thehumanized TRA-8 prepared in this Example 26 are adjusted to have theconcentration of the final product of interest of 100 ng/ml withRPMI1640 medium containing 10% FCS by estimating their concentrations inthe fluids according to the method described in Example 26. Each of thesolutions of the expression products thus adjusted to 100 ng/ml is usedto produce serial dilutions by repeating serial 2-fold dilution withRPMI 1640 containing 10% FCS. Each of the diluted humanized TRA-8solution (H1L1, H2L2, H2L3, H2L4, H3L3, H3L4 or H4L5) is added to eachwell at 50 μl per well. After reacting at 37′C for 12 hours, 50 All of25 μM PMS containing 1 mg/ml XTT is added (final concentrations of 250μg/ml for XTT and 5 μM for PMS). After incubating for 3 hours, theabsorbance at 450 nm of each well is measured to calculate the cellviability by using the reduction ability of mitochondria as the index.

The viability of the cells in each well is calculated according to thefollowing formula:Viability (%)=100×(a−b)/(c−b)wherein “a” is the measurement of a test well, “b” is the measurement ofa well with no cells, and “c” is the measurement of a well with noantibody added.

As a result, the tested humanized antibodies are demonstrated to induceapoptosis in cells of T lymphoma cell line expressing human DR5 antigen.

Furthermore, the apoptosis-inducing activity of humanized TRA-8 to PC-3is examined by adding taxol according to the method described in Example25.

Human prostate cancer cell line PC-3 (ATCC No. CRL-1435) is obtainedfrom American Tissue Culture Collection (ATCC) and maintained in F-12KNutrient Mixture (21127-022, Gibco BRL) containing 10% fetal bovineserum (FBS, Hyclone), 1% L-Glutamine-200 mM (25030-149, Gibco BRL) and0.5% Penicillin Streptomycin Solution (P-7539, Sigma). RPMI1640 medium(MED-008, IWAKI) supplemented with 10% FBS and 0.5% PenicillinStreptomycin Solution is used in the following experiment. Exponentiallygrowing PC-3 cells are collected by trypsinization and washed twice withfresh medium. The cells are then counted, resuspended in fresh medium ata density of 5×10⁴ cells/ml and distributed in triplicate intoflat-bottomed 96 well plates (3598, Corning-Coster) in a total volume of100 l/well one day before the start of the experiment. A representativeanti-cancer drug, Paclitaxel (169-18611, Wako) dissolved indimethylsulfoxide (10 mg/ml) is diluted in fresh medium and then addedto the 96-well plates containing the cells at 50 μl/well. The finalconcentrations of dimethylsulfoxide are less than 0.1%. After incubationfor 24 hr at 37° C. in 5% CO₂ atmosphere, humanized TRA-8 antibody(H1L1, H2L2, H2L3, H2L4, H3L2, H3L3, H3L4 or H4L5) diluted in freshmedium is added to the wells. After incubation for a further 24 hr, 50μl of Minimum Essential Medium (11095-098, Gibco BRL) containing 1 mg/mlof XTT and 25 mM of PMS is added to the wells and the plates areincubated for 6 hr. OD450 is then measured by ARVO HTS 1420 MultilabelCounter (Wallac Berthold) and the cell viability is calculated asfollows.Cell viability (%)=(OD450 for the well containing cells treated withTaxol and/or TRA-8 (agent(s))−OD450 for the well containing neithercells nor agent)×100/(OD450 for the well containing cells with noagent−OD450 for the well containing neither cells nor agent)

As a result, the tested humanized antibodies are demonstrated to induceapoptosis in human prostate cancer cells expressing human DR5 antigen.

Any patents or publications mentioned in the specification areindicative of the level of those skilled in the art to which theinvention pertains. These patents and publications are hereinincorporated by reference to the same extent as if each individualpublication was specifically and individually indicated to beincorporated by reference.

The present invention is not limited in scope by the above-referenceddeposit or the embodiments disclosed in the examples which are intendedas illustrations of a few aspects of the invention and any embodimentswhich are functionally equivalent are within the scope of thisinvention. Various modifications of the invention in addition to thoseshown and described herein will become apparent to those skilled in theart and are intended to fall within the scope of the appended claims.

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What is claimed is:
 1. A purified antibody having the same epitopespecificity as a humanized version of a monoclonal antibody produced bymouse-mouse hybridoma TRA-8 having ATCC Accession Number PTA-1428. 2.The antibody of claim 1, wherein the antibody comprises a heavy chaincomprising a constant region of a human immunoglobulin G1 (IgG1) heavychain.
 3. The antibody of claim 1, wherein the antibody comprises alight chain comprising a constant region of a human immunoglobulin kappalight chain.
 4. The antibody of claim 1, wherein the heavy chain of theantibody is encoded by a nucleotide sequence with up to 25% variation ascompared to the nucleotide sequence encoding SEQ ID NO:56, and whereinthe heavy chain has the same CDRs as a humanized version of a monoclonalantibody produced by mouse-mouse hybridoma TRA-8 having ATCC AccessionNumber PTA-1428.
 5. The antibody of claim 1, wherein the light chain ofthe antibody is encoded by a nucleotide sequence with up to 25%variation as compared to the nucleotide sequence encoding SEQ ID NO:72,and wherein the light chain has the same CDRs as a humanized version ofa monoclonal antibody produced by mouse-mouse hybridoma TRA-8 havingATCC Accession Number PTA-1428.
 6. The antibody of claim 1, wherein theantibody is linked to a ligand.
 7. The antibody of claim 1, wherein theantibody comprises a heavy chain comprising a framework regioncomprising amino acid residues 107-119 of SEQ ID NO:56.
 8. The antibodyof claim 1, wherein the antibody comprises a light chain comprising aframework region comprising amino acid residues 100-212 of SEQ ID NO:72.9. A pharmaceutical composition comprising the antibody of claim 1 and apharmaceutically acceptable carrier.
 10. The composition of claim 9,further comprising an additional therapeutic agent.
 11. The compositionof claim 10, wherein the additional therapeutic agent is an anti-cancercompound.
 12. A commercial kit comprising the antibody of claim 1,packaged in a suitable container with instructions for use.
 13. The kitof claim 12, wherein the container further comprises an additionaltherapeutic agent.
 14. The kit of claim 13, wherein the additionaltherapeutic agent is an anti-cancer compound.
 15. A purified antibodyhaving the same epitope specificity as an antibody comprising a heavychain comprising a variable region comprises SEQ ID NO:56 and a lightchain comprising a variable region comprising amino acid residues 1-108of SEQ ID NO:72.
 16. The antibody of claim 15, wherein the heavy chaincomprises a constant region of a human immunoglobulin G1 (IgG1) heavychain.
 17. The antibody of claim 15, wherein the light chain comprises aconstant region of a human immunoglobulin kappa light chain.
 18. Apharmaceutical composition comprising the antibody of claim 15 and apharmaceutically acceptable carrier.
 19. The composition of claim 18,further comprising an additional therapeutic agent.
 20. The compositionof claim 19, wherein the additional therapeutic agent is an anti-cancercompound.
 21. A commercial kit comprising the antibody of claim 15,packaged in a suitable container with instructions for use.
 22. The kitof claim 21, wherein the kit further comprises an additional therapeuticagent.
 23. The kit of claim 21, wherein the additional therapeutic agentis an anti-cancer compound.